JP2005245331A - Device for forming thin film, thin film device and method for producing the film device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for forming a thin film on the basis of a quite new principle, that can keep excellent stability of the thin film without any influence of artificial manipulation, a thin film device and a method for producing the film device. <P>SOLUTION: The film is formed by covering micropores formed on a base plate. At this time, a hole-closing phenomenon with a solvent is utilized to form the thin film. In other words, a solution formed by dissolving the materials for forming the thin film into a solvent is fed on the base plate having the micropores to form the thin film so as to close the micropores by swelling the film with the solvent. Then, the solvent is vaporized to open the micropores and to extend the formed thin film. The thin film is, for example, lipid film. The base plate is of a silicone resin, for example, of a polydimethylsiloxane. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a thin film forming device for forming a self-assembled thin film such as a planar lipid film that can be used for functional analysis of a cell membrane, for example, and further formed using such a thin film forming device. The present invention relates to a thin film device and a manufacturing method thereof.

  In order to analyze the molecular functions that occur in the cell membrane, it is necessary to observe in detail the behavior of remarkable proteins, but the elements that make up the actual cell membrane are extremely complex, and analysis focusing on a single function It will be very difficult.

  Therefore, an artificial membrane model that artificially constructs a lipid bilayer membrane, which is the simplest component of a cell membrane, and inserts only a specific protein for analysis is proposed. Artificial membrane models can be divided into two types: a liposome model having a closed sphere like a cell and a planar lipid membrane model in which a lipid membrane is formed planarly in micropores.

  Liposomes are molecular aggregates in which lipid molecules spontaneously form in water and have a bilayer structure in which lipid molecules are arranged two-dimensionally. In general, it can be easily prepared in a flask, and membrane-inserted proteins and peptides can be incorporated into the liposome membrane. However, when analyzing the function and characteristics of the inserted membrane protein, reaction detection in the liposome is an indirect method using a fluorescent label or the like.

  On the other hand, a model of a planar lipid membrane is formed using micropores. This model system has the advantage that the composition of the two components can be arbitrarily set across the lipid membrane, and the movement of the substance that permeates the membrane can be analyzed. In particular, since it is possible to detect an ionic current sandwiching a lipid membrane and control a membrane potential, it is used as an experimental system that gives much information to analysis of ion channel functions related to nerve transmission. Naturally, research on these ion channels related to neurophysiology involves observations using actual cells, and there is a method for detecting ion currents that pass through cell membranes, called the patch clamp method, which is an electrophysiological technique. Generally done. However, in order to directly understand the detailed behavior of channel proteins and the interaction between molecules, it is considered that observation with a planar lipid membrane model system is effective.

  For example, in the report of Yanagida et al.'S research group in 2002, an artificial lipid planar membrane system was constructed on an agarose substrate in order to analyze the target ion channel protein at a single molecule level. We have succeeded in simultaneously measuring the structural changes caused by the binding of ligands electrically and optically.

  By the way, two methods called the painting method reported by Mueller et al. In 1962 and the bonding method reported by Montal et al. Yes (see Non-Patent Document 1). These membrane formation methods are the same in that a polytetrafluoroethylene sheet (so-called Teflon (registered trademark) sheet) in which micropores with a diameter of several hundreds μm are formed is used, except that the method for developing lipid molecules is different. .

In the painting method, lipid molecules are dispersed in a decane solvent, and the solution is applied with a brush or pipette so as to close the micropores. If it does so, it will be in the state where the lipid solution became the state pinched | interposed into the aqueous solution from the both sides of a micropore, and it is a principle that a lipid molecule spontaneously forms a bilayer by hydrophobic interaction. In addition, the laminating method is an application of a monomolecular film formation technique called the Langmuir-Blodgette method to planar film formation. First, lipid molecules are formed at the gas-liquid interface of two solutions sandwiching micropores. To form a monomolecular layer. Next, the interface of the monomolecular film is raised so that the monomolecular films on both sides are bonded together in the micropores. As a result, the target planar lipid bilayer is formed.
Okada Yasunobu Editor, "New Patch Clamp Experiment Method", p208-224, Yoshioka Shoten (2001)

  The procedure of these formation methods is a simple operation that can be performed manually, but because of its simplicity, the control of nano-level molecular membranes cannot but depend on the self-assembly of lipid molecules. The skill of the experimenter working on the reproducibility of formation is required. For example, as seen in the painting method, when applying lipid using a brush, the lipid component adhering to the micropores is very low depending on the shape and material of the brush used and the user's operation method. It is uniform and greatly affects the properties of the double layer structure formed. Formation of a monomolecular film in the bonding method is also an operation based on the speculation that it may be a monomolecular film, and does not precisely control the vibration of the interface and the interaction with the wall surface.

  The most important factor involved in the stability of the lipid membrane formed by these methods is the interaction between the shape of the micropores used in the experiment and the lipid molecules exhibited by the material surface. In general, polytetrafluoroethylene sheets exhibiting hydrophobicity are used. This is because they are stable with respect to the organic solvent used and for the purpose of orienting the hydrophobic portion of the lipid molecule to the substrate surface. is there.

  Furthermore, in the production of microholes, a method of producing microholes with sharp edges is used, such as drilling by electric discharge or scraping a hollow formed by pushing a stainless steel bar with an obtuse angle with a razor. ing. These micropore fabrication methods were proposed by individual experimenters after trial and error as methods that can be easily fabricated by laboratory-level machining techniques. It is not standardized as a common tool between the two.

  Therefore, the results of experiments using the planar lipid membrane model reported at present are compared to the extent to which the effect of variation due to the lipid membrane formation method used has affected the target phenomenon. It is difficult to judge.

  Therefore, mass production is possible, it is possible to produce more precise micropores, and there is a need for a more mechanized method for forming a planar lipid membrane with less manual operations such as brushing and pasting. .

  The present invention has been proposed in view of such conventional circumstances, and is intended for thin film formation based on a completely new principle, in which the stability of the thin film formed by an artificial operation or the like is not greatly affected. An object is to provide a device, a thin film device, and a manufacturing method thereof.

  Since the present inventors can quickly handle biomolecules and switch between liquid feeds according to the microflow operation, the lipid molecules are placed at a target location to move the interface of an aqueous solution that promotes film formation. It came to think that it might be effective. In addition, as a method for developing a lipid solution, a lipid membrane structure can be constructed in a microspace on the substrate by applying the principle that the substrate material itself is mechanically deformed to promote thinning of lipid molecules. I came to think that it might be. The present invention has been completed based on these ideas.

  That is, the thin film forming device of the present invention includes a substrate having micropores that swells with a solvent, and the thin film is formed so as to cover the micropores by closing the micropores due to the swelling. And

  In the thin film forming device of the present invention, the thin film is automatically formed so as to cover the micropores by the operation of swelling (closed pores) of the micropores with the solvent. Thereby, it is possible to arrange | position a thin film on a board | substrate planarly and in an array form. Further, the film to be formed is arranged so as to have two minute spaces with the film interposed therebetween.

  Further, the thin film device of the present invention is formed by covering a minute hole formed in the substrate, and the thin film is formed with the minute hole being closed and stretched by the opening of the minute hole. It is characterized by that. Furthermore, the thin film device manufacturing method of the present invention is a thin film device manufacturing method for forming a thin film by covering a micropore formed in a substrate, and the thin film device is formed by utilizing a micropore closing phenomenon caused by a solvent. It is characterized by forming.

  For example, a silicone resin (polydimethylsiloxane) used for microdevice fabrication has a property of reversibly swelling and shrinking with a certain organic solvent. When micropores are formed in a substrate of such a material and an organic solvent in which lipids are dissolved is supplied to the substrate, a micropore closing operation occurs due to swelling of the organic solvent. Thereafter, when the organic solvent is vaporized, the micropores change to the original shape, and the lipid layer formed on the micropores is stretched in the horizontal direction by the opening operation. Thereby, a membrane structure in which lipid molecules are configured as, for example, a bilayer membrane is constructed. The above operation is not caused by any artificial operation, and therefore, the stability of the thin film is not affected by an artificial factor or the like.

  The present invention performs thin film formation by utilizing the expansion characteristics of the material of the micropore array chip, and can construct a microscale space inside the planar film. Also, a large number of thin film arrays can be formed at one time. They can be formed simultaneously. In addition, the stability of the thin film is not affected by human factors.

  In this way, if a planar membrane-type thin film structure (such as a bilayer structure of lipid molecules) can be constructed in the micro space on the substrate, miniaturization to on-chip and integration with other micro analysis technologies It can be expected that the pharmacological analysis of the receptor using the artificial membrane model used in conventional biological membrane research can be applied as a biochip that can be performed continuously and with a very small amount of sample.

  Hereinafter, a thin film forming device, a thin film device, and a manufacturing method thereof to which the present invention is applied will be described in detail with reference to the drawings.

  Various artificial molecular membranes have been proposed so far, and much information has been given to understand the structure and functions of actual cell membranes. Among them, the bilayer structure formed by self-assembly of phospholipids, which are cell components, can be regarded as the most simplified artificial membrane model of the actual cell membrane. It has been helpful in understanding the phenomenon. For example, it has been reported that acetylcholine receptors that play important functions in neurophysiology can be isolated from power generation organs such as Sybilei and can express their channel function by inserting them into lipid membranes. It was.

  Thus, the construction of a model system in which only notable receptors are inserted into the membrane makes it possible to analyze the physicochemical phenomena of ion channels more clearly. Furthermore, it has become possible to observe direct pharmacological mechanisms of neurotransmitters and toxins that act on receptors. Thus, artificial membrane models using planar lipid membranes have contributed greatly to receptor research. Furthermore, in recent years, many attempts have been made to apply the superior molecular mechanisms of living organisms from an engineering point of view, and the principle of the formation of nanostructures formed by cell membranes has become the basis of research on biomaterials and molecular devices based on supramolecular structures. Greatly utilized in design. In particular, the function of a living body specifically recognizing molecules is extremely interesting, and many attempts have been made to apply it to biosensors. For example, researches are being conducted on taste sensors that follow the tastes of living organisms and blood glucose level sensors that utilize the selective catalytic action of enzymes.

  What should be noted here is that many of the intelligent functions of these biomolecules are phenomena that take place in the cell membrane. Therefore, in order to elucidate the mechanism of these molecular machines and apply them, it is considered that the major issue is how to construct the cell membrane environment artificially and stably build it as the basis of molecular devices.

  On the other hand, microscale patterning and three-dimensional molding can be performed on various substrates by a semiconductor microfabrication technology that has been remarkably developed in recent years. Attempts have been made to apply microstructures that can be produced by these processing techniques to various scientific fields. In particular, chemical reaction systems using microspaces can be made as small as 1/100 to 10,000 times less than the amount of reagents used in conventional experimental systems, and the substance diffusion effect and surface area associated with microscales. Since the effect of the above is remarkably reflected, research for proposing a novel solution reaction using these phenomena has also been conducted.

  Furthermore, miniaturization of analyzers for detecting reactions occurring in these microspaces is also progressing, and this is called μTAS (Micro Total Analysis system), which integrates conventional solution analysis processes on a chip of several centimeters square. Device development is drawing attention. These technologies are expected to be widely deployed as analytical technologies in the biotechnology field. The usefulness of these analytical devices is to reduce the amount of sample samples, shorten reaction and measurement times, and reduce the size of measurement systems. For example.

  The inventors of the present invention have also been researching a system for performing chemical analysis by efficiently arranging desired biomolecules by utilizing a microscale microstructure pattern. At that time, as a method for controlling the delivery of a minute amount of a solution sample, a solution delivery system using a microchannel structure has been studied. The present invention proposes a new method for forming a planar lipid membrane using such a microflow technique.

  The thin film forming device of the present invention performs thin film formation by utilizing the operation of regularly closing micropores. First, the principle of forming a thin film (for example, lipid membrane) based on the micropore closing operation will be described. The principle of thin film formation in the present invention is as shown in FIG.

  That is, as shown in FIG. 1 (a), a slide glass 1 on which a substrate (silicone resin film) 2 having a minute hole 3 is formed is prepared, and as shown in FIG. A solution 4 in which a thin film forming material is dissolved in a solvent, for example, a hexane solution of lipid (Phosphatidylcholine) is dropped. Then, the silicone resin film as the substrate 2 swells in the direction of the arrow in the figure due to the swelling action by the solvent (hexane), and the closing operation of the micropores 3 occurs. Thereafter, the solvent is immediately vaporized at room temperature, whereby a thin thin film 5 made of only a thin film forming material is formed on the surface of the silicone resin film 2 as shown in FIG. At the same time, the micropores 3 change to the original shape as the solvent evaporates. As shown in FIG. 1D, the thin film 5 formed on the top surface of the micropores 3 is moved horizontally by the opening operation. Stretched. Thereby, for example, it can be approximated to a bilayer structure formed by lipid molecules.

  Although the thin film device of the present invention is formed by the above principle, the thin film forming material constituting the thin film 5 is not limited to the lipid, and any material can be used as long as it is a molecule capable of molecular orientation. . However, lipid is preferable from the viewpoint of artificially constructing the environment of the cell membrane. The thin film 5 is assumed to be a bimolecular film, but is not limited thereto, and may be a monomolecular film or the like.

  The material constituting the substrate 2 is optimally a silicone resin film as described above. However, the material is not limited to this as long as it can swell with a solvent and perform a micropore closing operation. . Therefore, it may be appropriately selected depending on the combination with the solvent to be used. For example, when hexane, which is one kind of organic solvent, is used as the solvent as described above, a silicone resin or the like is used as a material constituting the substrate 2. In addition, as a silicone resin, the resin material which consists of polydimethylsiloxane can be mentioned, for example.

  The substrate 2 has microholes 3 formed therein. For example, the microholes 3 are arranged in an array so that a large number of thin film devices can be collectively formed. Although it is the dimension of the micropore 3, it is preferable that the diameters of a hole are 20 micrometers-100 micrometers, for example. If the diameter of the minute hole 3 exceeds the above range, the closing operation may be difficult. On the contrary, even if it falls below the above range, smooth closing operation may be difficult, and the space constituted by the micro holes 3 may be too small, and there may be restrictions in use. The depth of the microhole 3 may be about 40 μm to 100 μm. However, in order to smoothly perform the closing operation, it is preferable to increase the depth of the microhole 3 as the hole diameter increases.

  The microhole 3 is preferably a through-hole penetrating the substrate 2, but is not necessarily limited thereto, and the surface opposite to the surface on which the thin film is formed may be blocked. However, in that case, there is a possibility that the use of the space formed by the minute holes 3 may be restricted.

  The micro holes 3 can be easily formed by, for example, a photolithography technique. Specifically, a resist pattern corresponding to the minute holes is formed by photolithography, and silicone resin or the like is poured using this resist pattern as a mold. Then, the resist pattern portion is transferred to the substrate 2 as a minute hole.

  The thin film 5 formed on the upper surface of the minute hole 3 is stretched by opening the minute hole. Therefore, it is preferable that the substrate 2 and the thin film 5 are firmly bonded to some extent. From such a viewpoint, it is also effective to perform a surface treatment on the surface of the substrate 2 (thin film forming surface). The surface treatment may be any surface treatment that can improve the adhesion between the substrate 2 and the thin film 5, and examples thereof include surface treatment with a silane coupling agent and oxygen plasma treatment. As the silane coupling agent used for the surface treatment, a silane coupling agent having an amino group is highly effective.

  As described above, when the thin film device of the present invention is manufactured, the thin film 5 is formed so as to cover the micro holes 3 formed in the substrate 2 by utilizing the phenomenon of the micro holes 3 being closed by the solvent. That is, a solution 4 in which a thin film forming material is dissolved in a solvent is supplied onto the substrate 2 on which the micropores 3 are formed, and film formation is performed in a state where the micropores 3 are closed by swelling of the solvent. The minute holes 3 are opened by evaporation, and the formed thin film 5 is stretched.

  At this time, the organic solvent is usually used as the solvent. However, the present invention is not limited to this. For example, depending on the type of the thin film forming material, an aqueous solvent may be used.

  In the production method, basically, the molecular orientation of the thin film forming material (molecule) is performed by utilizing the affinity of the hydrophobic group and hydrophilic group of the thin film forming material with the solvent or air. Can be incorporated into a micro flow system to form a more automated thin film and control molecular orientation. In this case, a flow channel in which the solution passes through the micropores 3 is constructed, and solution feeding, organic solvent removal, and aqueous solution injection necessary for thin film formation are continuously performed.

  Even when a microflow channel is used, a thin film can be formed in the same manner, but the principle is as shown in FIG. In this case, the second slide glass 6 is arranged on the substrate 2 with a minute interval to form a slit. Then, this slit is used as a microflow channel. First, as shown in FIG. 2A, a solution 4 (solution in which lipid is dissolved in an organic solvent) 4 is dropped onto the open end of the microflow channel. Then, the solution 4 is fed into the flow path by the capillary effect and flows through the upper surface of the micropore 3. At the same time, the silicone resin swells with an organic solvent (for example, hexane), and the micropores 3 are closed. As the organic solvent evaporates, the gas-liquid interface advances through the flow path, the surface of the micropores 3 is thinly coated with thin film forming material molecules (lipid molecules), and the thin film 5 is disposed on the upper surface of the micropores 3 and thinned. Is done. Next, water 7 is dropped on the open end of the microflow channel. Then, the water 7 is also fed into the flow path by the capillary effect, and flows on the upper surface of the micropore 3, that is, on the thin film 5. As a result, the hydrophilic group side of the thin film-forming molecule is oriented toward the flowing water, and self-organization is performed. In this case, since the liquid feeding operation is performed by a capillary phenomenon, the thin film forming operation only requires dropping of the solution, and can be extremely simplified.

  Hereinafter, the present invention will be described based on specific experimental results.

(Preparation of micropores by photolithography technology)
The silicon wafer was cut into 3 × 4 cm squares and subjected to ultrasonic cleaning with ultrapure water for 5 minutes. Next, the silicon wafer was immersed in an acetone solution and boiled and washed for 5 minutes. Further, it was immersed in a hydrofluoric acid solution [hydrofluoric acid (10 ml) / ammonium fluoride (60 ml)] for 5 minutes to remove the oxide film on the wafer surface, washed with ultrapure water, and then dried with nitrogen gas. Photoresist (SU8-50) was dropped onto this silicon wafer, and developed with an arbitrary film thickness by a spin coater. This was prebaked with a hot plate at 95 ° C. for 30 minutes. Next, an easy mask pattern was exposed to UV with a mask aligner. Then, it post-baked with the hotplate on the conditions of 95 degreeC and 30 min. This was immersed in a SU8 developer, and a mold having a concavo-convex pattern of photoresist on a silicon wafer could be produced.

  On the other hand, the trade name Silpot184 (manufactured by Dow Corning) and the trade name Catalysts Silpot184 (manufactured by Dow Corning) were mixed at 10: 1 to obtain a PDMS solution. It was degassed using a desiccator for 10 minutes. About 40 mg of this PDMS was poured into the previously prepared SU8 mold, a slide glass was placed on the upper surface, and the whole was fixed with a clamp. This was heated at 80 ° C. for 1.5 hours. After heating, the mixture was allowed to stand until it reached room temperature, and the solidified PDMS resin was taken out from the mold. At this time, the PDMS sheet is firmly attached to the slide glass, and is a convenient lamination for supporting the PDMS sheet having a thickness of several tens of micrometers. As described above, a PDMS sheet laminated on a slide glass was used as an array chip.

  The above process is schematically shown in FIGS. In order to form a mold, as shown in FIG. 3A, a photoresist is dropped on the silicon wafer 11 to form a resist layer 12. Then, UV exposure is performed through the mask pattern 13. By this UV exposure, as shown in FIG. 3B, the resist layer 12a in the portion irradiated with UV light is cured. If this is developed, a template in which the resist layer 12a remains is formed as shown in FIG. Next, as shown in FIG. 4A, the PDMS 14 is poured onto the mold. If it peels off from a casting_mold | template after hardening, as shown in FIG.4 (b), the micropore 14a will be formed in PDMS14. FIG. 5 shows an example of forming a micro uneven pattern (micropores 14 a) on the PDMS 14. The micro holes 14a are formed with a diameter of a μm, a depth of d μm, and an interval of L μm.

(Examination of conditions for the closing process by the shape of the micropores)
In accordance with the manufacturing process described above, a PDMS chip having a diameter of 20 μm and a thickness of 40 to 60 μm was manufactured. When a hexane solution is dropped onto the surface of the chip, structural changes such as opening [FIG. 6A] and closing hole [FIG. 6B] are observed as schematically shown in FIG. This is probably because the hexane solution penetrates PDMS and is temporarily swollen. This swelling was found to return immediately to its original shape as the hexane solution evaporated. Therefore, as conditions for such PDMS micropores to undergo structural changes, pay attention to the size of the diameters of several micropores and the thickness of the PDMS sheet, etc. went.

  First, conditions based on the size of the micropores and the sheet thickness were examined. PDMS sheets having arbitrary thicknesses (corresponding to a depth d) of 40 to 200 μm are prepared for the micropore size a = 10 μm, 20 μm, 50 μm, 100 μm, and 200 μm, respectively, and accompanying the dropping of hexane. Observation was made on the swelling behavior of the micropores. The result is shown in FIG. From this, it was found that the thickness (depth d) of the PDMS sheet proportional to the size a of the diameter of the micropores is necessary for the micropores to completely change the closed pore structure. For example, when a micropore array having a diameter of 100 μm was produced, when the PDMS thickness was 40 μm and 60 μm, the expansion was insufficient and the micropores could not be completely closed. Further, in the case of micropores having a diameter of 200 μm, even when the PDMS thickness was 200 μm, the pores swelled but the pores did not completely occur.

  Next, conditions were examined based on the size of the micropores and the spacing between the holes. PDMS sheets having a pattern of hole arrangement intervals L = 10 μm, 20 μm, 50 μm, 100 μm, and 200 μm are prepared for the micropore size a = 10 μm, 20 μm, 50 μm, 100 μm, and 200 μm, respectively, and hexane is dropped. Observations were made on the behavior of the micropore swelling associated with. The result is shown in FIG. In these observations, it is found that when the distance between the micropores is too large, the interaction between the micropores does not occur, concentric swelling occurs individually, and the lattice-like pore closing operation does not occur. It was. For example, in the case of 100 μm micropores, the mutual micropores act at intervals smaller than 100 μm, whereas when they are separated by 200 μm, the mutual action does not occur.

(Formation of lipid membranes utilizing PDMS micropore closing phenomenon)
An operation based on the principle shown in FIG. 1 was performed using a micro-hole array chip having a diameter of 20 μm, which was a micro-hole pattern in which a micro-hole closing operation occurred, and observation was performed with a microscope. As a result, first, immediately after the lipid solution was dropped, the micropores were immediately closed in a lattice pattern. Thereafter, hexane vaporization began, the interface of the lipid solution became smaller, and as the interface moved, lipid molecules were thinly coated. As soon as hexane was completely vaporized, the closed micropores were opened, and the lipid film coated on the top surface of the closed pores was stretched to form a thin film showing a light interference pattern. The results are shown in FIG.

  The interference pattern generated in the thin film changed its color in a fluid manner, and after a few minutes, all of the formed thin film was ruptured. These interference patterns correspond to the direction of the closed micropores. Horizontal stripes were observed in the vertically closed micropores, and vertical stripes were observed in the horizontally closed micropores (FIG. 10). reference). These interference patterns of light are a phenomenon that occurs in a submicron thin film, and it is known that each color of the interference pattern corresponds to the formed film thickness. From this, it is considered that the thin film structure of lipid molecules generated in these micropores has a film thickness gradient in a direction perpendicular to the direction of the interference fringes. As the formation mechanism, as shown in FIG. 11, it is considered that the swelling is small in the end portion of the closed pore, and the structural change is considered to occur largely in the central portion, and the effect of stretching the lipid membrane is generated accordingly. It is guessed.

(Examination of lipid membrane formation by surface treatment of PDMS)
The lipid membrane formed in previous experiments was found to be very unstable and rupture within minutes. Therefore, in order to make the formed lipid membrane more stable, an attempt was made to modify a functional group that enhances the adsorption with lipid molecules on the PDMS surface. Specifically, the amino group was modified on the PDMS surface with a silane coupling agent, and the effect was examined. In addition, as a surface modification of PDMS, the effect of introducing silanol groups by oxygen plasma was also compared.

  In modifying the amino group on the surface of PDMS, 3-aminopropyltriethoxysilane (APTES: 3-Aminopropyltriethoxysilane, manufactured by Chisso Corporation) was used. Its chemical formula is as shown in Chemical Formula 1.

Moreover, the following apparatus was used when performing oxygen plasma treatment on the PDMS surface.
Reactive ion etching equipment (SAMCO, RIE-10R) Reaction conditions: 100 scm, 200 W, O ₂ gas used, 9.0 Pa, 10 sec

  First, as a comparison of surface treatment to PDMS, Table 1 shows the measurement results of contact angles before treatment, after oxygen plasma treatment, and after amino group modification. In the case of introducing an amino group, the contact angle hardly changed compared to before the treatment. Further, after the oxygen plasma treatment, the surface becomes hydrophilic, but it is known that the hydrophilic effect is lost to the original state as time passes. Therefore, in the subsequent experiment, observation was performed using a sample 3 hours after the oxygen plasma treatment.

  Next, using the micropore chip having a different micropore pattern and surface treatment, the same lipid membrane formation operation as in the previous section was performed, and the observation results are summarized in Table 2.

  When the micropore diameter was 10 μm, the stretch of the lipid membrane was small, and no thin film was formed. In addition, in a micropore having a diameter of 100 μm, the lipid membrane is stretched, but because of the large operation, the thin film was immediately broken. On the other hand, it was found that a thin film having a pore diameter of 20 μm can form a thin film for the stretching operation, and in particular, when an amino group is modified, thin films having various colors as shown in FIG. 12 are formed. . In this case, as shown in FIG. 12 (a), immediately after the micropores are opened, the film starts to be slightly colored near the center of the film, and gradually spreads as a flat surface as shown in FIG. 12 (b). I was able to observe how it went. The color exhibited by these thin films is considered to be a light interference pattern peculiar to the thin films. Furthermore, since the interference pattern was monochromatic, it was suggested that the structure was very uniform and submicron thin.

  Next, the details of the surface structure of the produced film were observed using a scanning electron microscope (SEM). The result is shown in FIG. The spherically expanded structure as shown in FIG. 13A is considered to be a structure in which the thin film formed on the upper surface of the micropore is stretched by high vacuum. Further, it can be confirmed that the formed thin film of lipid is arranged so as to cover the upper surface of the micropore. Furthermore, from the enlarged view shown in FIG. 13 (b), broken film fragments could be confirmed, suggesting that these films were very thin films of submicrons. FIG. 13C shows the formation state of the lipid thin film inferred from the observation results of FIGS. 13A and 13B. It is presumed that the lipid thin film 22 is formed on the microarray chip 21 in the state as shown in FIG.

  In the above experiment, when the PDMS surface was modified with an amino group and an array chip having a micropore diameter of 20 μm was used, a plurality of lipid membrane structures shown in FIG. 12 were formed at the same time. Since these thin films exhibit various colors, it is considered that films having different thicknesses are formed. It was confirmed that a lipid film having a black portion was formed therein (see FIG. 14). In general, it is known that the double layer structure formed by lipid molecules has a thickness of 10 nm or less, does not cause light interference, and appears black. Therefore, it is considered that a double-layer structure of lipid molecules is formed in the black portion confirmed this time.

(Formation of planar lipid membrane by micro flow system)
A PDMS micropattern (micropore array) was prepared according to the same procedure as described above. Then, as shown in FIG. 15, a chip 32 having a flow path structure was bonded to the upper surface of the microhole array 31 and the both surfaces were sandwiched between slide glasses 33 and 34 to conduct an experiment.

  The observation of the film formation process is shown in FIG. First, the lipid solution was dropped into the flow channel injection portion (the introduction portion of the microchannel). Then, as shown in FIG. 16B, the lipid solution permeated into the flow channel due to the capillary phenomenon. And the micropore which touched the solution caused a structural change due to swelling and closed in a lattice shape. After several tens of seconds, hexane in the flow channel gradually vaporized from the end of the flow channel, and the volume of the solution in the flow channel decreased as shown in FIG. And in the part which the hexane solution vaporized, as shown in FIG.16 (d), opening operation | movement of a micropore started and the lipid membrane was extended on the micropore upper surface.

  Thereafter, a lipid membrane having the same interference pattern as that observed in the previous section “Formation of lipid membrane using PDMS micropore closing phenomenon” could be formed. These membranes ruptured within minutes. Therefore, when an aqueous solution was sent to the membrane surface in order to stabilize the lipid membrane after the membrane was formed, a black foam-like structure was formed as shown in FIG. Thus, when the film | membrane formed on the micropore upper surface was set | placed in aqueous solution, the disappearance process occurred. In the atmosphere, the thin film that had an interference pattern appears black in an aqueous solution, so the following possibilities are considered. One is that the refractive index of the aqueous solution on the upper surface of the film is different from that of the air inside the film. Another possibility is that the membrane is broken and the air in the micropores is stacked as bubbles, and the air gradually absorbs into the PDMS. This is because PDMS has the property of being very permeable to gas.

It is a schematic diagram explaining the basic principle of film formation in this invention. It is a schematic diagram explaining the formation principle of the lipid membrane by a flow formation system. It is a schematic diagram which shows the preparation process of the casting_mold | template by a photolithographic technique, (a) is an exposure process, (b) is a hardening state, (c) shows the casting_mold | template formation state by image development. It is a schematic diagram which shows formation of the micro uneven | corrugated pattern by transcription | transfer of a casting_mold | template, (a) is a PDMS pouring state, (b) shows the formed uneven pattern. It is a schematic perspective view which shows the formed micropore array. It is a schematic diagram which shows the open state (a) of the micropore by hexane dripping, and the closed state (b). It is a figure which shows the relationship between the magnitude | size of a micropore, the thickness of a sheet | seat (depth of a micropore), and closing operation | movement. It is a figure which shows the relationship between the magnitude | size and space | interval of a micropore, and closing operation | movement. It is a photograph of the lipid molecule thin film formed on the micropores. It is a photograph which shows a response | compatibility with the interference pattern of light and the direction of a closed hole in a lipid molecule thin film. It is a schematic diagram which shows the cross-sectional structure of the film | membrane estimated from the interference pattern of light. It is a photograph which shows the mode of film formation at the time of amino group modification, (a) shows a mode immediately after micropore opening, (b) shows a mode after 5 minutes. It is a SEM observation image of the formed lipid membrane, (a) is a 400 times SEM image, (b) is a 1000 times SEM image, (c) is a cross-sectional prediction figure. It is a photograph which shows the black area | region formed in the lipid membrane. It is a schematic perspective view which shows the structure of the chip | tip used for formation of the planar lipid membrane by a micro flow system. It is a photograph which shows the formation process of the lipid membrane by a micro flow system.

Explanation of symbols

1 slide glass, 2 substrate (silicone resin), 3 micropores, 4 solution, 5 thin film, 11 silicon wafer, 12 resist layer, 13 mask pattern, 14 PDMS, 14a micropore, 21 microarray chip, 22 lipid thin film, 31 Micropore array, 32 channel structure chip, 33, 34 slide glass

Claims (35)

  A device for forming a thin film, comprising: a substrate having micropores that is swollen by a solvent; and a thin film is formed so as to cover the micropores by closing the micropores due to the swelling.   2. The thin film forming device according to claim 1, wherein the micropores are arranged in an array.   3. The thin film forming device according to claim 1, wherein the material constituting the substrate is a silicone resin.   4. The thin film forming device according to claim 3, wherein the silicone resin is made of polydimethylsiloxane.   5. The thin film forming device according to claim 1, wherein the surface of the substrate is surface-treated.   6. The device for forming a thin film according to claim 5, wherein the surface of the substrate is surface-treated with a silane coupling agent.   The thin film forming device according to claim 6, wherein the silane coupling agent has an amino group.   6. The device for forming a thin film according to claim 5, wherein the surface of the substrate is surface-treated by oxygen plasma treatment.   The device for forming a thin film according to any one of claims 1 to 7, wherein a diameter of the micropore is 20 µm to 100 µm.   The device for forming a thin film according to claim 9, wherein the depth of the micropore is 40 μm to 100 μm.   The device for forming a thin film according to claim 1, wherein the micro hole is a through hole penetrating the substrate. A thin film is formed covering the micropores formed in the substrate,
The thin film is formed in a state where the micropores are closed, and is stretched by opening the micropores.   The thin film device according to claim 12, wherein spaces are formed on both sides of the thin film.   The thin film device according to claim 12 or 13, wherein the thin film is a lipid film.   The thin film device according to claim 12, wherein the thin film is a molecularly oriented monomolecular film or a bimolecular film.   The thin film device according to claim 12, wherein the material constituting the substrate is swollen by a solvent.   The thin film device according to claim 16, wherein the material constituting the substrate is a silicone resin.   The thin film device according to claim 17, wherein the silicone resin is made of polydimethylsiloxane.   The thin film device according to claim 12, wherein the substrate surface is surface-treated.   The thin film device according to claim 19, wherein the substrate surface is surface-treated with a silane coupling agent.   The thin film device according to claim 20, wherein the silane coupling agent has an amino group.   The thin film device according to claim 19, wherein the substrate surface is surface-treated by oxygen plasma treatment.   The thin film device according to any one of claims 12 to 22, wherein a diameter of the micropore is 20 µm to 100 µm.   24. The thin film device according to claim 23, wherein the depth of the micropore is 40 [mu] m to 100 [mu] m.   25. The thin film device according to claim 12, wherein the micro hole is a through hole penetrating the substrate. A method of manufacturing a thin film device that covers a micropore formed in a substrate to form a thin film,
A method of manufacturing a thin film device, wherein the thin film is formed by utilizing a phenomenon of micropores closed by a solvent.   A solution in which a thin film forming material is dissolved in a solvent is supplied onto a substrate on which micropores are formed, and film formation is performed in a state where the micropores are closed by swelling of the solvent, and then the micropores are formed by evaporation of the solvent. 27. The method of manufacturing a thin film device according to claim 26, wherein the thin film is formed by stretching the formed thin film.   28. The method of manufacturing a thin film device according to claim 27, wherein lipid is used as the thin film forming material and an organic solvent is used as the solvent.   29. The method of manufacturing a thin film device according to claim 28, wherein an aqueous solution is injected onto the thin film after the thin film is formed.   30. The method of manufacturing a thin film device according to claim 29, wherein a slit is formed on a thin film forming surface of the substrate, and the organic solvent and the aqueous solution are injected using the slit.   31. The method of manufacturing a thin film device according to claim 26, wherein a surface treatment is performed on the substrate before forming the thin film.   32. The method of manufacturing a thin film device according to claim 31, wherein the surface treatment is performed with a silane coupling agent.   32. The method of manufacturing a thin film device according to claim 31, wherein the surface treatment is performed by oxygen plasma.   34. The method of manufacturing a thin film device according to claim 26, wherein the microhole is formed by a photolithography technique.   35. The method of manufacturing a thin film device according to claim 34, wherein the micro holes are formed by forming a resist pattern corresponding to the micro holes by a photolithography technique and transferring the resist pattern to a substrate.

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