JP2015133174A - Transmission electron microscope and liquid observation environment cell device for localized plasmon resonance expression and manufacturing method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liquid observation environment cell device, which allows for high resolution observation when used in a transmission electron microscope, and expresses localized plasmon resonance, at a low manufacturing cost.SOLUTION: A liquid observation environment cell C consists of a lower sealing layer 1 of 10 nm thick composed of carbon, an adhesive layer 2 of 10 nm thick composed of ceramics, a metal layer 3 of 10 nm thick for adsorbing a molecule M, and an adhesive layer 4 of 10 nm thick composed of ceramics. In order to express plasmon resonance, the metal layer 3 is composed of Au, Ag, Cu or Al, and the diameter D of the opening OP in the adhesive layer 2, metal layer 3 and adhesive layer 4 is from several tens nm to 500 nm ((A) of Fig. 3). The molecule M is adsorbed to the wall surface of the metal layer 3. On the adhesive layer 4, an upper sealing layer 5 of 10 nm thick composed of carbon is formed ((B) of Fig. 3). An electron beam EB proceeds from the upper sealing layer 5 toward the lower sealing layer 1, and a TEM image is obtained. Cross-sectional observation of solid (wall surface of the metal layer 3)/liquid interface, and cross-sectional observation of the molecule M are possible ((C) of Fig. 3).

Description

  The present invention relates to a transmission electron microscope (TEM), a liquid observation environment cell (EC) device for expressing localized plasmon resonance, and a manufacturing method thereof.

  In the fields of development of biomaterials / pharmaceuticals, understanding of tissue growth mechanisms in living organisms, improvement of lubrication performance, etc., it is important to elucidate the structure of the solid / liquid interface, the morphology of the molecules adsorbed on the interface, and the behavior. .

  The observation of the solid-liquid interface and the adsorbed molecules at the interface has recently been performed by a transmission electron microscope using a container called a liquid observation environment cell (EC). In other words, by immersing only the vicinity of the adsorbed molecule that is the sample in the liquid observation environment cell, the sample is cut off from the vacuum, and the scattering distance of the electron beam is suppressed by minimizing the distance of the liquid through which the electron beam passes. ing.

  The first conventional liquid observation environment cell is incorporated in the sample holder. This liquid observation environment cell can introduce different liquids with the sample between the upper and lower sealing layers, and can also be used for electrochemical measurements and the like (see Patent Document 1).

  In the second conventional liquid observation environment cell, a liquid is contained in a sample itself, and the sample is put in a general-purpose holder. Since this liquid observation environment cell uses a general-purpose holder, it is excellent in convenience.

Special table 2013-535795 gazette

Seung-Man Yang et al., “Nanomachining by Colloidal Lithography”, Colloidal Lithography, small 2006, 2, No.4, pp.458-475

  However, in the first conventional liquid observation environment cell described above, since the liquid observation environment cell is unique, if the liquid observation environment cell deteriorates or breaks down, it must be replaced together with the sample holder. There is a problem that the cost becomes high. In addition, since the distance between the upper and lower sealing layers of the sample is mechanically maintained by an O-ring or the like, the distance between the upper and lower sealing layers is increased, thus increasing the thickness of the liquid observation environment cell. There is a problem that the scattered absorption of the line becomes large and high-resolution observation is impossible. Furthermore, since the liquid observation environment cell has almost no wall surface, most of the sample (adsorbed molecules) is adsorbed vertically to the upper and lower sealing layers, and the vertical direction of the solid (sealing layer) / liquid interface and the longitudinal direction of the adsorbed molecules. However, there is a problem that it is impossible to observe the cross section of the solid (sealing layer) / liquid interface and the cross section of the adsorbed molecules in the short direction.

  The above-described second conventional liquid observation environment cell cannot sufficiently control the thickness and size of the sample (molecule). Therefore, the cross-section observation of the solid / liquid interface and the cross-section observation of the adsorbed molecule are also possible. There is a problem that it is impossible.

  Furthermore, since the above-described first and second conventional liquid observation environment cells do not have a metal layer, there is a problem that it is impossible to observe the adsorption process of molecules by plasmon resonance detection. That is, there is a problem that it is impossible to use the liquid observation environment cell in combination with observation using a transmission electron microscope and observation using localized plasmon resonance.

  In order to solve the above-mentioned problems, a transmission electron microscope and a localized plasmon resonance expression liquid observation environment cell apparatus according to the present invention include a lower sealing layer made of carbon having a thickness of nanometer order, A metal layer having a thickness on the order of nanometers disposed on the lower sealing layer, and a plurality of openings having a size on the order of nanometers formed at least partially regularly in the metal layer. Thereby, a large number of liquid observation environment cells having a small thickness and a wide wall surface are provided. Further, by accommodating the liquid in the opening and adsorbing the molecule to the metal layer which is the wall surface of the opening, it is possible to observe the cross section of the solid (particularly the wall surface) / liquid interface and the cross section of the adsorbed molecule in the short direction. Furthermore, thin metal layers that are at least partially separated by regular openings exhibit localized surface plasmon resonance.

  In addition, the manufacturing method of the transmission electron microscope and the localized plasmon resonance expression liquid observation environment cell device according to the present invention includes a step of forming a sacrificial layer on a substrate, and a carbon having a thickness on the order of nanometers on the sacrificial layer. Forming a lower sealing layer comprising: at least partially regularly depositing a plurality of colloids having a size on the order of nanometers on the lower sealing layer; and a thickness on the order of nanometers on the colloid and the sacrificial layer The step of forming the metal layer, the step of removing the colloid after the formation of the metal layer, and the step of peeling off the substrate by removing the sacrificial layer after the removal of the colloid are provided.

  According to the present invention, even if a part of the liquid observation environment cell is deteriorated or destroyed, it is not necessary to replace it, and therefore the manufacturing cost can be reduced. In addition, since the thickness of the liquid observation environment cell is reduced, the scattering absorption of the electron beam is reduced, and high-resolution observation is possible. Furthermore, since the wall surface becomes wider, cross-sectional observation of the solid (wall surface) / liquid interface and cross-sectional observation of adsorbed molecules in the short direction are possible. Furthermore, the refractive index (or dielectric constant) of adsorbed molecules can be observed by the expression of localized plasmon resonance, and therefore the adsorption process can be observed.

1 is a top view showing an embodiment of a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression according to the present invention. FIG. FIG. 2 is an electron micrograph showing a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression in FIG. 1, (A) is a scanning electron microscope (SEM) photo, and (B) is a higher magnification than (A). (C) is a transmission electron microscope (TEM) photograph at a higher magnification than (B). FIG. 2 is a cross-sectional view of the liquid observation environment cell of FIG. 1, (A) is a state before enclosing liquid and molecules, (B) is a state after enclosing liquids and molecules, and (C) is placed in a transmission electron microscope. The placed state is shown. It is sectional drawing for demonstrating the manufacturing method of the transmission electron microscope of FIG. 1, and the liquid observation environment cell apparatus for localized plasmon resonance expression. 2 is an in situ TEM image obtained with a transmission electron microscope using the transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression of FIG. It is a graph which shows the absorption spectrum characteristic of the liquid electron observation environment cell apparatus for a transmission electron microscope and localized plasmon resonance expression of FIG. FIG. 2 is a timing diagram showing an in-situ localized plasmon resonance peak value shift amount of the transmission electron microscope and the liquid observing environmental cell device for expressing localized plasmon resonance of FIG. 1.

  FIG. 1 is a top view showing an embodiment of a transmission electron microscope and localized plasmon resonance expression liquid observation environment cell apparatus according to the present invention, and FIG. 2 is a transmission electron microscope and localized plasmon resonance expression liquid of FIG. It is an electron micrograph which shows an observation environment cell apparatus, (A) is a scanning electron microscope (SEM) photograph, (B) is a SEM photograph of higher magnification than (A), (C) is higher magnification of (B). It is a transmission electron microscope (TEM) photograph. In FIGS. 1 and 2, the upper sealing layer described later is not formed, and neither liquid nor adsorbed molecules exist.

  As shown in FIG. 1, the transmission electron microscope and the localized plasmon resonance-expressing liquid observation environment cell apparatus have a plurality of liquid observation environment cells C at least in a regular range R, that is, partially regularly arranged. Yes. For example, as shown in FIG. 2 (A), a transmission electron microscope having a size of 50 μm × 50 μm and a liquid observation environment cell device for local plasmon resonance expression are used in the numbers shown in FIG. 2 (B) and (C). 100 to several thousand liquid observation environment cells C having a size of 100 nm × 100 nm are arranged.

  3 is a cross-sectional view of the liquid observation environment cell C of FIG. 1, in which (A) is a state before enclosing the liquid and molecules, (B) is a state after enclosing the liquid and molecules, and (C) is a transmission electron. The state mounted in the microscope is shown.

  As shown in FIG. 3A, the liquid observation environment cell C before enclosing the liquid and the molecules has a thickness of about 5 to 500 nm, for example, 10 nm, made of carbon, and a thickness of about ceramic, for example, AlN. An adhesive layer 2 having a thickness of 5 to 500 nm, for example 10 nm, a metal layer 3 having a thickness of about 5 to 500 nm, for example 10 nm, for adsorbing molecules, and an adhesive layer 4 having a thickness of about 5 to 500 nm, for example 10 nm, made of ceramics such as AlN. In this case, the adhesive layer 2 has an adhesive action between the lower sealing layer 1 and the metal layer 3, and the adhesive layer 4 has an adhesive action between an upper sealing layer 5 and the metal layer 3 described later. Accordingly, the adhesive layer 2 can be omitted when the adhesive action between the lower sealing layer 1 and the metal layer 3 is strong, and the adhesive layer 4 when the adhesive action between the upper sealing layer 5 and the metal layer 3 is strong. Can be omitted. The adhesive layers 2 and 4 also have an effect of stabilizing the metal layer 3.

  In FIG. 3A, the metal layer 3 is made of, for example, gold (Au), silver (Ag), copper (Cu), or aluminum (Al) in order to express the localized plasmon resonance described later. The diameter D of the opening OP of the adhesive layer 2, the metal layer 3, and the adhesive layer 4 is 5 to 500 nm. In this case, further, the metal layer 3 is selected from the above-described metals having good adsorptivity with the molecule M described later.

  3B, the liquid L and the molecule M are sealed in the openings OP of the adhesive layer 2, the metal layer 3 and the adhesive layer 4 in FIG. Adsorb to the wall. Thereafter, an upper sealing layer 5 made of carbon and having a thickness of about 5 to 500 nm, for example, 10 nm, is formed on the adhesive layer 4.

  In FIG. 3C, the electron beam EB travels from the upper sealing layer 5 toward the lower sealing layer 1. As a result, a TEM image is obtained. In this case, since the electron beam EB passes through the cross section of the solid, particularly the wall surface of the metal layer 3 and the liquid L, and the cross section of the molecule M, the cross section of the solid (wall surface of the metal layer 3) / liquid interface is observed in the TEM image. In addition, cross-sectional observation of molecule M can be performed.

  Next, a method for manufacturing the transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression in FIG. 1 will be described with reference to FIG.

  First, referring to FIG. 4A, a sacrificial layer 102 made of, for example, aluminum is formed on a substrate 101 made of silicon or glass by sputtering, vapor deposition, or the like. Here, the substrate 101 is for ensuring the flatness of the lower sealing layer 1 which will be described later, and when the substrate 101 and the lower sealing layer 1 are directly bonded, the lower sealing layer 1 is difficult to peel off. Is interposed between the substrate 101 and the lower sealing layer 1.

  Next, referring to FIG. 4B, a carbon layer as the lower sealing layer 1 is formed on the sacrificial layer 102 by sputtering, vapor deposition, chemical vapor deposition (CVD), molecular beam epitaxial (MBE). It is deposited by the spraying method, etc.

  Next, referring to FIG. 4C, colloids 103 having the same size are partially and regularly deposited. The colloid 103 is a metal of 5 to 500 nm, an organic material, an inorganic material, or a biological material, and is made of, for example, polystyrene as the organic material. The size and distribution of the colloid 103 can be adjusted by the solution concentration, deposition time, ionic strength, etc. (see Non-Patent Document 2), but since the colloid 103 is normally charged, the distribution is shown in FIG. Can be regular in regular range R. The colloid 103 may be regularly arranged in the entire range.

  Next, although not shown, the size of the colloid 103 is reduced according to the diameter D of the opening OP by an etching method as necessary. Examples of the etching method include a reactive ion etching (RIE) method, a plasma etching method, an ultraviolet irradiation method, a chemical etching method, and an ozone irradiation method.

  Next, referring to FIG. 4D, a 10 nm thick adhesive layer 2 made of ceramic, for example, AlN, a 10 nm thick metal layer 3 made of, for example, Au, and a 10 nm thick adhesive layer 4 made of ceramic, for example, AlN. Are sequentially formed by sputtering, vapor deposition or the like.

  Next, referring to FIG. 4E, the colloid 103 is removed by, for example, a physical lift-off method. Thus, an opening OP having a diameter D is formed by the adhesive layer 2, the metal layer 3, and the adhesive layer 4.

  Next, referring to FIG. 4F, the glass substrate 101 is physically peeled by removing the sacrificial layer 102 by an etching method.

  At this stage, the transmission electron microscope and the localized plasmon resonance expression liquid observation environment cell apparatus of FIG. 1 are shipped to the user, and the following steps (G) and (H) of FIG. 4 are executed by the user. It will be.

  Referring to FIG. 4G, the transmission electron microscope and the localized plasmon resonance expression liquid observation environment cell device shown in FIG. Adsorbed on the wall surface of the metal layer 3.

  Finally, referring to FIG. 4H, the transmission electron microscope and the grid are moved and pulled up from the liquid L while adhering the 10-nm thick upper sealing layer 5 made of carbon floating in the liquid L. A liquid observation environment cell device for expressing localized plasmon resonance is completed.

  FIG. 5 is an in situ TEM image obtained with a transmission electron microscope using the transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression of FIG. The liquid L in FIG. 5 is a gold colloid solution, and the metal layer 3 is made of Au. As shown in (A), (B), (C), and (D) of FIG. 5, changes in Au are observed.

  FIG. 6 is a graph showing the absorption spectrum characteristics of the transmission electron microscope and the local plasmon resonance expression liquid observation environment cell device of FIG. That is, according to the absorption spectrum of visible light and infrared light having a three-layer structure including the adhesive layer 2, the metal layer 3, and the adhesive layer 4, peak values P1, P2, and P3 indicating the expression of localized plasmon resonance are generated. ing. Here, I is a case where a three-layer structure exists in the air, II is a case where the three-layer structure is immersed in water, and III is a case where the upper part of the three-layer structure is also immersed in water. Thus, since the metal layer 3 having a thickness on the order of nanometers is partially separated by the openings OP formed at least partially regularly, the peak values P1, P2, P3 occurs.

  FIG. 7 is a timing chart showing the in-situ localized plasmon resonance peak value shift amount of the transmission electron microscope and the liquid observing environmental cell device for expressing localized plasmon resonance of FIG. In FIG. 7, when the protein avidin is put together with water into a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression, and then the water is removed, the local plasmon resonance peak value shift amount changes. Was recognized. This shift amount is adsorbed on a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression and shows a refractive index (or dielectric constant) corresponding to the adsorption amount of protein avidin. Therefore, the adsorption process of protein avidin can be observed.

  In addition, the nanometer order in this invention means 5-500 nm.

  Further, the present invention can be applied with any modification within the obvious range of the above-described embodiment.

1. . . Lower sealing layer . . 2. Adhesive layer . . Metal layer 4. . . 4. Adhesive layer . . Upper sealing layer 101. . . Substrate 102. . . Sacrificial layer
C. . . Liquid observation environment cell
R. . . Regular scope
OP. . . Opening
L. . . liquid
M. . . molecule

Claims (8)

A lower sealing layer made of nanometer-thick carbon,
A metal layer having a thickness on the order of nanometers disposed on the lower sealing layer,
A transmission electron microscope and a liquid observation environment cell device for expressing localized plasmon resonance, wherein a plurality of openings having a size on the order of nanometers are at least partially regularly formed in the metal layer. further,
A first adhesive layer having a thickness on the order of nanometers disposed between the lower sealing layer and the metal layer;
A second adhesive layer having a thickness on the order of nanometers disposed on the metal layer;
The transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression according to claim 1, wherein the first and second adhesive layers are formed with openings that communicate with the openings of the metal layer.   2. The transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression according to claim 1, wherein the metal layer is made of any one of gold, silver, copper, and aluminum.   The transmission electron microscope and the liquid observation environment cell device for local plasmon resonance expression according to claim 2, wherein the first and second adhesive layers are made of ceramics. Forming a sacrificial layer on the substrate;
Forming a lower sealing layer of carbon having a thickness on the order of nanometers on the sacrificial layer;
Depositing a plurality of colloids on the order of nanometers on the lower sealing layer at least partially regularly;
Forming a metal layer with a thickness on the order of nanometers on the colloid and the sacrificial layer;
Removing the colloid after forming the metal layer;
A method of manufacturing a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression, comprising: removing the sacrificial layer after removing the colloid; and removing the substrate. further,
Forming a first adhesion layer having a thickness on the order of nanometers on the colloid and the sacrificial layer before forming the metal layer;
Forming a second adhesive layer having a thickness on the order of nanometers after the formation of the metal layer and before the removal of the colloid. 6. Transmission electron microscope and localized plasmon resonance expression according to claim 5 Of manufacturing a liquid observation environment cell device for medical use.   6. The method of manufacturing a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression according to claim 5, wherein the metal layer is made of any one of gold, silver, copper, and aluminum. The method of manufacturing a transmission electron microscope and a liquid observation environment cell device for local plasmon resonance expression according to claim 6, wherein the first and second adhesive layers are made of ceramics.