JP7179281B2 - Nanotweezers, Nanotweezers Intermediate, Tweezers Kit, Method for Producing Nanotweezers, and Method for Producing Nanotweezers Intermediate - Google Patents

Description

TECHNICAL FIELD The present invention relates to nanotweezers, nanotweezer intermediates, coating agents, tweezers kits, methods for producing nanotweezers, and methods for producing nanotweezer intermediates.

Conventionally, nanotweezers for handling micron-order samples have been proposed (see, for example, Patent Document 1). The nanotweezers have a pair of arms and an actuator that drives them formed by semiconductor microfabrication technology, and gripping and releasing of the sample is performed by opening and closing the arms with the actuator.

For example, when handling cells in a liquid, the tip of the arm is immersed in the liquid to grasp the cell, so the liquid adheres to the arm, and impurities in the liquid and foreign matter adhering to the liquid become dirt. There is a problem that it remains on the arm surface. Therefore, in the nanotweezers described in Patent Document 1, a water-repellent chemisorptive monomolecular film is formed on the surface of the arm to suppress the adhesion of the liquid, thereby preventing the arm from being soiled.

JP 2008-110436 A

However, when a cell is grasped by the arm, the cell adheres to the surface of the arm and the grasped cell cannot be released from the arm.

A nanotweezer according to a first aspect of the present invention has a pair of arms that can be freely opened and closed, and a drive unit that performs opening and closing operations of the pair of arms, and each of the arms has a zwitterionic structure. A gripping portion is provided on which a coating layer of a molecularly oriented monomolecular film is formed, a metal film is formed on at least a surface of a substrate of the gripping portion on which the coating layer is formed, and the coating layer is formed by the molecule having the zwitterionic structure containing an atom that binds to the metal of the metal film at the end, and the atom binding to the metal of the metal film, or the zwitterionic structure is formed by bonding with the metal film via a surface initiator containing atoms that bond with the metal of the metal film.
A nanotweezers manufacturing method according to a second aspect of the present invention includes preparing a nanotweezers intermediate body having a pair of openable and closable arms having a grip portion and a driving portion for opening and closing the pair of arms, and A coating agent that forms a metal film on at least the surface of the base material of the gripping portion of the nanotweezers intermediate on which the coating layer is formed, and contains a molecule having a zwitterionic structure containing a sulfur atom that binds to the metal film, A coating layer of an oriented monomolecular film of molecules having the zwitterionic structure is formed by coating the surface of the metal film of the grip portion.
A nanotweezers intermediate according to a third aspect of the present invention is a method for producing a nanotweezers intermediate for producing the nanotweezers according to the first aspect, comprising: a pair of freely openable and closable arms having a grip; a driving unit for opening and closing the pair of arms, wherein a metal film is formed on the surface of the base material of the gripping unit, and the surface of the metal film has an orientation having the zwitterionic structure. It is coated with a surface initiator to form a monomolecular coating layer.
A method for producing a nanotweezers intermediate according to a fourth aspect of the present invention is a method for producing a nanotweezers intermediate for producing the nanotweezers according to the first aspect, comprising a pair of openable and closable grips having a grip portion A nanotweezer having an arm and a driving unit for opening and closing the pair of arms, and a metal film formed on the substrate surface of the gripping unit is prepared, and polymerization of a monomer having a zwitterionic structure is initiated. A surface initiator for is prepared, and the surface of the metal film is coated with the surface initiator.
A nanotweezers manufacturing method according to a fifth aspect of the present invention includes preparing the nanotweezers intermediate according to the above aspect, applying a coating agent containing a molecule having a zwitterionic structure to the gripping portion, and using the surface initiator. A step of forming a coating layer of an oriented monomolecular film having the zwitterionic structure on the coated surface of the grip portion starting from the surface initiator.
A tweezers kit according to a sixth aspect of the present invention is a coating agent for forming the coating layer of the nanotweezers of the first aspect, the coating agent containing a molecule having a zwitterionic structure; and a driving unit for opening and closing the pair of arms, a metal film is formed on the surface of the base material of the gripping unit, and the surface of the metal film is and a nanotweezer intermediate coated with a surface initiator for forming a coating layer of the oriented monolayer having the zwitterionic structure.

Advantageous Effects of Invention According to the present invention, it is possible to provide nanotweezers that suppress non-specific adsorption of an object to be gripped to the nanotweezers and that have a gripping portion that has a high water retention capacity. Biological samples and the like can be handled appropriately with this nanotweezers.

FIG. 1 shows an embodiment of nanotweezers. FIG. 2 is a diagram showing the details of the grip. FIG. 3 is a diagram showing an example of a coating agent used for the coating layer. FIG. 4 is a diagram illustrating an example of handling of a biological sample using nanotweezers. FIG. 5 is a diagram illustrating the effect of suppressing nonspecific adsorption by coating with zwitterionic molecules. FIG. 6 shows the molecular structures of MPC, SBMA and CBMA. FIG. 7 shows a variation of the zwitterionic structure. FIG. 8 shows another variation of the zwitterionic structure. FIG. 9 is a diagram showing the morphology of a coating layer having a zwitterionic structure. FIG. 10 is a diagram showing the arrangement of positive and negative charges in a zwitterionic structure. FIG. 11 shows three products in MPC-SH synthesis. FIG. 12 shows three products in SBMA-SH synthesis. FIG. 13 shows the synthesis procedure of surface initiator (BiBOE) ₂ S ₂ . FIG. 14 shows a PSBMA formed with a surface initiator (BiBOE) ₂ S ₂ .

Embodiments for carrying out the present invention will be described below with reference to the drawings. FIG. 1 is a diagram showing one embodiment of the nanotweezers, and is a plan view of the nanotweezers 1. FIG. The nanotweezers 1 shown in FIG. 1 are integrally manufactured from, for example, an SOI (Silicon on Insulator) wafer using a semiconductor microfabrication technique. An SOI wafer is obtained by forming a SiO ₂ layer on one of two Si single crystal plates and bonding them together via the SiO ₂ layer.

The nanotweezers 1 include a pair of arms 13a and 13b, a pair of electrostatic actuators 14a and 14b, a pair of support portions 17a and 17b, a pair of connecting portions 18a and 18b, a pair of arm support portions 19a and 19b, and a base 11. I have. The electrostatic actuator 14a is provided with a fixed electrode 15a and a movable electrode 16a, and the electrostatic actuator 14b is provided with a fixed electrode 15b and a movable electrode 16b.

The electrostatic actuator 14a for the symmetrical arm 13a and the electrostatic actuator 14b for the arm 13b have the same structure. The fixed electrode 15a and the movable electrode 16a both have a comb-teeth shape, and are opposed to each other in such a manner that the comb-teeth are engaged with each other with a gap therebetween. The fixed electrode 15 a is formed on the base 11 . On the other hand, the movable electrode 16a is elastically fixed to the pedestal 11 by a thin beam-shaped support portion 17a. The fixed electrode 15b and the movable electrode 16b also have the same structure.

The arms 13a and 13b are elastically fixed to the pedestal 11 via thin beam-like arm support portions 19a and 19b, respectively. The arm 13a and the movable electrode 16a are connected by a connecting portion 18a, and the arm 13b and the movable electrode 16b are connected by a connecting portion 18b.

A terminal 12c is connected to the left arm 13a in the drawing, a terminal 12a is connected to the fixed electrode 15a, and a terminal 12b is connected to the movable electrode 16a. On the other hand, a terminal 12d is connected to the arm 13b on the right side of the drawing, a terminal 12e is connected to the movable electrode 16b, and a terminal 12f is connected to the fixed electrode 15b. Terminals 12a, 12b, 12e, 12f, and 12g are terminals for applying voltage. The terminals 12c and 12d are provided for detecting the amount of electricity acting between the arms 13a and 13b. Therefore, the arms 13a and 13b are insulated from the movable electrodes 16a and 16b, respectively. A terminal 12g is a ground terminal for preventing the pedestal 11 from becoming a floating electrode.

When a voltage is applied between the fixed electrode 15a and the movable electrode 16a, the movable electrode 16a moves toward the fixed electrode 15a (to the right in the drawing), causing the arm 13a to move toward the arm 13b (to the right in the drawing). driven. When the voltage application is released, the arm 13a returns to its original position. The movement of the right arm 13b is only reversed. Similarly, the movable electrode 16b moves toward the fixed electrode 15b (leftward in the drawing), causing the arm 13b to approach the arm 13a (leftward in the drawing). driven by As a result, a minute object can be gripped between the gripping portions 130a and 130b, and the gripped minute object can be released.

A manufacturing method for forming the nanotweezers 1 using an SOI (silicon on insulator) substrate is well known (for example, Japanese Unexamined Patent Application Publication No. 2006-26825, etc.), so description thereof will be omitted here. As the substrate used for forming the nanotweezers 1, in addition to the SOI substrate, a substrate having a single crystal silicon layer on a glass substrate, a substrate having an SOI layer on an amorphous silicon substrate or a polysilicon substrate, or the like can be used. . In either case, arms 13a, 13b, etc. are formed in the silicon layer. In an embodiment, the base material of arms 13a, 13b is silicon.

FIG. 2 is a diagram showing the details of grips 130a and 130b formed at the tip portions of arms 13a and 13b. The thickness of the tip portions of the arms 13a and 13b is reduced stepwise to form gripping portions 130a and 130b (hatched portions) shaped as shown in FIG. As will be described later, at least the surfaces of the gripping portions 130a and 130b of the arms 13a and 13b are coated with a zwitterionic structure. In the specification, the coating may also be referred to as a coating layer.

For example, the length L of gripping portions 130a and 130b for gripping a biological sample such as cells is set longer than the sample, and the width W and thickness t are set to be approximately the same as the size of the sample. As an example of the dimensions of the gripping portions 130a and 130b, the length L is 100 μm, the thickness t is 1 to 25 μm, and the width W is 1 to 30 μm.

[Coating agent for forming coating layer]
Next, a coating agent for forming a coating layer on the grip portions 130a and 130b will be described. In this embodiment, a coating layer having a zwitterionic structure is formed on the surfaces of grips 130a and 130b. This coating layer is formed to improve handling performance when handling biological samples such as cells with the nanotweezers 1 .

FIG. 3 is a diagram showing an example of a coating agent used as a coating layer. The coating agent 300 uses 2-methacryloyloxyethylphosphorylcholine (MPC) having a thiol group (SH group) at its terminal. Hereinafter, 2-methacryloyloxyethylphosphorylcholine (MPC) having a thiol group (SH group) at its end is referred to as MPC-SH. Since MPC-SH is known to have a high bonding strength with Au (gold), a gold film 301 is formed on the surfaces of the grips 130a and 130b in this example. In the MPC-SH shown in FIG. 3, the portion indicated by reference numeral 310 corresponds to negative ions, and the portion indicated by reference numeral 320 corresponds to positive ions. A water molecule binds to this zwitterionic structure portion by electrostatic action, resulting in a hydrated structure.
Molecules (monomers and polymers) having such a zwitterionic structure are hereinafter referred to as zwitterionic molecules (zwitterionic monomers and zwitterionic polymers). Therefore, the coating layer having the zwitterionic structure is a film containing zwitterionic molecules on the substrate surfaces of the grips 130a and 130b.

When the grips 130a and 130b on which the gold film 301 is formed are immersed in a solution containing MPC-SH, SH groups of MPC-SH react with the gold film 301 to form Au--S bonds. The MPC-SH bound on the gold film 301 spontaneously forms a monomolecular layer with high orientation due to intermolecular attractive interaction. This monomolecular layer is the covering layer 330 . Such a monolayer is called a self-assembled monolayer (SAM).

When the gold film is formed on the surfaces of the gripping portions 130a and 130b, for example, if the gold film is formed on the surface of the gripping portions after the chromium film is formed, the gold film is formed on the gripping portions directly. The film is difficult to peel off. Also, since S (sulfur) of the SH group strongly bonds with Au (gold), the metal film formed on the surfaces of the gripping portions 130a and 130b is preferably a gold film, but the metal film is necessarily limited to gold. Nickel or the like may be used instead. A thiol group (SH group) can also be used as a binding functional group when a nickel film is used as the metal film. In addition, although a thiol group (SH group) was used as the terminal binding functional group, a zwitterion molecule with a terminal configuration that corresponds to the material of the gripping surface may be used.

FIG. 4 is a diagram illustrating an example of handling of a biological sample by the nanotweezers 1. FIG. The example shown in FIG. 4 shows the case of transporting a cell 281 in a container 28 to another container 29 using the nanotweezers 1 . A buffer solution 280 is contained in the container 28 and the cells 281 are immersed in the buffer solution 280 . A buffer solution 280 is also contained in the container 29 of the transport destination.

First, the arms 13a and 13b of the nanotweezers 1 are moved directly above the container 28 in an open state. Next, the nanotweezers 1 are lowered downward in the figure and moved so that the cell 281 is positioned between the grasping portions 130a and 130b of the arms 13a and 13b. After that, the arms 13a and 13b are closed and the cell 281 is grasped by the grasping portions 130a and 130b. After grasping the cell 281 , the nanotweezers 1 are lifted upward in the drawing to let the cell 281 out of the buffer solution 280 in the container 28 into the air, and then transported into the buffer solution 280 in the container 29 . When the cell 281 gripped by the gripping parts 130a and 130b has been transported into the buffer solution 280 of the container 29, the arms 13a and 13b are opened to release the cell 281 into the buffer solution 280. FIG.

By the way, the inventors have discovered the following problems when transporting a biological sample using nanotweezers. When a water-repellent film is formed on the surface of the arm as in conventional nanotweezers, hydrophobic samples such as cells adhere to the surface of the gripping portion due to non-specific adsorption, and even if the gripping portions 130a and 130b are opened. It was found that the biological sample could not be released. Although it is not a phenomenon at the gripping part of nanotweezers, nonspecific adsorption of biological samples to the substrate surface can be suppressed by forming polyethylene glycol or zwitterion molecules on the substrate surface. It has been known.

FIG. 5 is a diagram for explaining the effect of suppressing nonspecific adsorption by coating with zwitterionic molecules. FIG. 5(a) is a diagram explaining the mechanism by which the protein 50 is adsorbed to the substrate 51 by non-specific adsorption. Counterions with opposite charges are attracted to the surface of charged substances, and water molecules are hydrated around them. Sodium ions 52 having opposite charges are bound on a negatively charged hydrophilic substrate 51, and water molecules 53 are hydrated around them. Also, sodium ions 52 are bound to the negatively charged portion of the protein 50, chloride ions 54 are bound to the positively charged portion, and water molecules 53 are hydrated around them. When the positively charged portion of protein 50 approaches the surface of the substrate (negatively charged), water molecules 53 are expelled together with the counterions (sodium ion 52, chloride ion 54) bound to each surface, resulting in the substrate Protein 50 is non-specifically adsorbed to the surface of 51 .

On the other hand, in FIG. 5(b), the surface of the substrate 51 is covered with the zwitterion molecules 55 constituting the coating layer 330, and the positively charged and negatively charged portions of the zwitterion molecules 55 are hydrated with the water molecules 53. take structure. In this case, even if the protein 50 similar to the case of FIG. 5A is close to the substrate surface, exchange of counter ions does not occur, so that the water molecules 53 can be stably hydrated on the substrate surface. As a result, non-specific adsorption of protein 50 is suppressed. Such an effect of suppressing nonspecific adsorption can be obtained whether the zwitterion molecule 55 is a polymer or a monomer.

In the case of polyethylene glycol, oxygen atoms are regularly arranged between long chains of hydrocarbons, and water molecules are hydrated around polyethylene glycol through hydrogen bonding. Therefore, it has the effect of suppressing non-specific adsorption as in the case of zwitterionic molecules. The present inventors coated the gripping portions 130a and 130b of the nanotweezers 1 in the case of polyethylene glycol and zwitterion molecules, respectively, and confirmed that the biological sample was easily released from the gripping portions 130a and 130b. did.

By the way, the inventor found that transporting a biological sample using nanotweezers poses a new problem other than non-specific adsorption. As shown in FIG. 4, when the cells 281 are transported from the container 28 to another container 29, the grasped cells 281 must once be released from the buffer solution 280 into the air. At that time, if there is no moisture around the cells 281, the cells 281 will die, so it is necessary to prevent the cells 281 from becoming dry. That is, it is essential to transport the cells 281 in a state where the buffer solution 280 is present around them.

As described with reference to FIG. 2, since the gripping portions 130a and 130b of the nanotweezers 1 have a microstructure, it is difficult to hold the buffer solution 80 in the portions of the gripping portions 130a and 130b that grip the cells 281 . That is, when the gripping portions 130a and 130b are pulled out of the buffer solution 280, the buffer solution 280 in the gripping portions 130a and 130b is taken to the buffer liquid surface side in the container 28 and remains on the gripping portion side. is considered to be impossible.

When polyethylene glycol was used as a coating agent for the coating layer of the gripping portions 130a and 130b, non-specific adsorption could be suppressed and release was possible. There was a problem that the buffer solution 280 was not sufficiently retained around the grasped cells 281 when released into the air, and the cells 281 dried up and died. On the other hand, when the coating layer 330 having the zwitterionic structure is formed on the gripping portions 130a and 130b as shown in FIG. I was able to prevent 281 from dying.

Both polyethylene glycol and zwitterion molecules have a hydrated structure with water molecules. It differs in that it has a hydrated structure due to its action. Electrostatic interactions are stronger than hydrogen bonds between water molecules, so zwitterionic molecules have a stronger attraction to water molecules. As a result, the effect of suppressing non-specific adsorption and the water retention amount (buffer retention amount) when the gripping portions 130a and 130b are exposed to the air from the buffer 280 are also improved by coating the zwitterion molecules. It is considered better to Thus, by coating the gripping portions 130a and 130b of the nanotweezers 1 with zwitterion molecules, it is possible to provide the nanotweezers 1 with excellent biological sample handling performance (release characteristics and moisture retention characteristics).

As described with reference to FIG. 2, the width W and thickness t of the gripping portions 130a and 130b are set to be approximately the same as the sample size, and the length L is set longer than the sample. When the sample is gripped by the gripping portions 130a and 130b, the sample is gripped by the tip portions of the gripping portions 130a and 130b as shown in FIG. At that time, the gripping portions 130a and 130b having the length L are not necessarily immersed in the buffer solution 280 entirely. In the present embodiment, the entire gripping portions 130a and 130b of the length L are coated, but it is not necessary to coat the entire length L. A coating may be applied to a range of about L/2 from the tip of the part. Zwitterion molecules may be coated only on a pair of opposing surfaces that grip cells without coating the entire gripping portion with zwitterion molecules.

Although MPC is used as the zwitterionic molecule in the above-described embodiments, various zwitterionic molecules other than MPC can be used as the coating agent. Typical zwitterionic monomers include SBMA (sulfobetaine methacrylate) and CBMA (Carboxybetaine methacrylate) as shown in FIG. 6, in addition to MPC.

There are many variations of the zwitterionic structure in the molecule, one example of which is shown in FIGS. The effect of suppressing nonspecific adsorption is not uniform, and differs depending on the structure of the zwitterion. I to XXIV in FIGS. 7 and 8 are as follows.
・I and XIV are ammoniophosphates (phosphobetaines or lecithin analogues).
- II, IV and XV are ammoniophosphonates (phosphonobetaines).
・III is ammoniophosphinates (phosphinobetaines).
・V and XVI are ammoniosulfonates (sulfobetaines).
- VI and XVII are ammoniosulfates.
- VII, X, XI, XVIII and XXI are ammoniocarboxylates (carbo- or carboxybetaines).
・VIII is ammoniosulfonamides.
・IX is ammonia-sulfon-imides.
・X is guanidiniocarboxylates (asparagineanalogs).
・XI is pyridiniocarboxylates.
・XII is ammonio(alkoxy)dicyanoethenolates.
・XIII is an ammoniaboronate.
・XIX is sulfoniocarboxylates.
・XX is phosphoniosulfonates.
・XXI is phosphoniocarboxylates.
• XXII is squareine dyes.
• XXIII and XXIV are oxypyridine betaines.

FIG. 9 is a diagram showing the morphology of a coating layer having a zwitterionic structure. In FIG. 9, (a) and (b) show the case where a coating layer with a zwitterionic structure is formed using a monomer, and (c) and (d) show a case where a coating layer with a polymer with a zwitterionic structure is formed. indicates when FIG. 9(a) shows the case of coating with a zwitterionic monomer such as MPC, which has zwitterions in each molecule. In the example shown in FIG. 9A, the positive ions electrically interact with the negative ions of adjacent molecules, resulting in a bent molecule structure.

FIG. 9(b) shows the case of coating a substrate surface with a combination of positively charged molecules and negatively charged molecules, and the positive and negative charges are distributed almost evenly over the entire surface. In this case, a positively charged molecule and a negatively charged molecule form a pair of zwitterion-like structures, and such a configuration is also referred to as a zwitterion structure in the present specification. When forming a zwitterionic structure having such a configuration, for example, positively charged monomers include 2-aminoethyl methacrylate (AEMA), 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA), 2-( methacryloyloxy)ethyl trimethylammonium chloride (MTAC), (3-acrylamidopropyl) trimethylammonium chloride (APTAC), etc. are used. (AMPS), methacrylic acid (MA), sodium methacrylate (NaMA), acrylic acid (AA), sodium acrylate (NaAA), etc. are used.

FIG. 9(c) shows a case where a zwitterionic monomer such as MPC is radically polymerized to form a polymer having a zwitterionic structure. ing. FIG. 9(d) shows a case where a positively charged monomer and a negatively charged monomer are combined and polymerized to form a zwitterionic structure. In zwitterionic polymers such as those shown in FIGS. 9(c) and (d), structures such as those shown in FIGS. 10A to 10K are possible with respect to the zwitterionic structure. 9(c) is for structure C, and FIG. 9(d) is for structure E. FIG. Any of the structures shown in FIG. 10 can provide the above-described effects (effect of suppressing non-specific adsorption and high water retention).

Further, when a coating layer having a zwitterionic structure containing a zwitterionic polymer is formed on the surface of the substrate, it is also possible to gel by cross-linking between linear polymer molecules. By making such a zwitterionic structure into a three-dimensional network structure, higher water retention is expected.
The types of coating layers formed on the gripping portions 130a and 130b can be confirmed by mass spectrometry, infrared spectroscopy, NMR, or the like.

(Example 1)
A method for synthesizing the coating agent (MPC-SH) and a procedure for coating when the nanotweezers 1 are coated with the MPC shown in FIG. 3 will be described.
(1A) Synthesis of MPC-SH 1.55 g (5.25 mmol) of MPC and 1.184 g (7.88 mmol) of 1,6-hexanedithiol were placed in a round-bottomed flask, and nitrogen gas was passed through it. 10.5 ml of chloroform deoxygenated by bubbling is added and stirred to dissolve both reagents (MPC and 1,6-hexanedithiol).
- Add 29.4 µl (0.2 mmol) of diisopropylamine to the above solution, and stir at room temperature for 24 hours under a nitrogen gas atmosphere.
- Add the reaction solution to an excess amount of acetone to produce a white precipitate, discard the supernatant, wash the white precipitate twice with acetone, and vacuum dry for 1 hour to dry.
・Add 100 ml of ultrapure water to the dried material to dissolve, and freeze-dry the dissolved material.
The products obtained by such a procedure are three kinds shown in (1) to (3) in FIG.

(1B) Coating procedure • The product synthesized in (1A) was dissolved in ultrapure water to prepare a 10 mM solution. Since the contents of the three types of products were unknown, the concentrations were calculated assuming that the products were all (1).
- The holding parts 130a and 130b of the nanotweezers 1 on which the gold film 301 is formed are immersed in the above solution for 1 to 2 minutes.
- After that, the grips 130a and 130b are immersed in ultrapure water for 1 minute and air-dried.
- Of the three types of products shown in FIG. 11, (1) MPC-SH and (2) MPC-SH dimer are self-organized on the surface of the gold film 301 formed on the gripping portions 130a and 130b. formed as an oxidized monolayer (SAM). The molecule (2) is obtained by dimerizing the thiol group portion of MPC-SH in (1) to create a double bond of S—S, and the S of the double bond portion of S—S is gold. It can be attached to the surface of membrane 301 .

(Example 2)
In Example 2, SBMA coating was performed in the same manner instead of MPC coating.
(2A) Synthesis of SBMA-SH 1.4 g (5 mmol) of SBMA and 1.125 g (7.5 mmol) of 1,6-hexanedithiol were placed in a round-bottomed flask and deoxygenated by bubbling nitrogen gas. 10.5 ml of distilled methanol is added and stirred to dissolve both reagents (SBMA and 1,6-hexanedithiol).
- Add 29.4 μl (0.2 mmol) of diisopropylamine to the above solution, and stir at room temperature for 24 hours under a nitrogen gas atmosphere.
- Add the reaction solution to an excess amount of acetone to produce a white precipitate, discard the supernatant, wash the white precipitate twice with acetone, and vacuum dry for 1 hour to dry.
・Add 100 ml of ultrapure water to the dried material to dissolve, and freeze-dry the dissolved material.
The products obtained by such a procedure are three kinds shown in (1) to (3) in FIG.

(2B) Coating procedure • The product synthesized in (2A) was dissolved in ultrapure water to prepare a 10 mM solution. Since the contents of the three types of products were unknown, the concentrations were calculated assuming that the products were all (1).
- The holding parts 130a and 130b of the nanotweezers 1 on which the gold film 301 is formed are immersed in the above solution for 1 to 2 minutes.
- After that, the grips 130a and 130b are immersed in ultrapure water for 1 minute and air-dried.
・Of the three types of products shown in FIG. 12, (1) SBMA-SH and (2) SBMA-SH dimer are self-organized on the surface of the gold film 301 formed on the gripping portions 130a and 130b. formed as an oxidized monolayer (SAM). The molecule (2) is a dimerized product in which the thiol group portion of SBMA-SH in (1) creates a double bond of S—S, and the S of the double bond portion of S—S is gold. It can be attached to the surface of membrane 301 .

(Example 3)
Example 3 describes a coating that forms a zwitterionic structural overlayer comprising a zwitterionic polymer. Here, the surface of the gold film 301 of the gripping portions 130a and 130b is coated with a surface initiator for synthesizing a polymer brush, and the surface initiator forms a zwitterionic polymer as a starting point.
Here, Bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide is used as an example of the surface initiator.

(3A) Synthesis of surface initiator (bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide) Bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide is shown in FIG. Bis(2-hydroxyethyl) disulfide is synthesized from 2-mercaptoethanol, and bis[2-(2 '-Bromoisobutyryloxy)ethyl]disulfide.

(3A-1) Synthesis of bis(2-hydroxyethyl)disulfide 7.8 g (100 mmol) of 2-mercaptoethanol is weighed and dissolved in 25 ml of ultrapure water.
• Dilute 8.5 ml of 30% H ₂ O ₂ solution with 10 ml of ultrapure water.
Add the above diluted H ₂ O ₂ solution while stirring the above 2-mercaptoethanol solution with a stirrer and stir at room temperature for 1 hour.
- Transfer the stirred solution to a separating funnel and extract with 50 ml of ethyl acetate three times.
- The above three ethyl acetate phases are put together and dehydrated with magnesium sulfate. After the magnesium sulfate is removed by filtration, the solvent is distilled off with a rotary evaporator.
・After that, dry overnight (approximately 16 hours) in a vacuum dryer.

(3A-2) Synthesis of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide ・Regarding bis(2-hydroxyethyl)disulfide synthesized in (3A-1), 3.09g was added to a 50ml tube. After weighing and adding 5.05 g of triethylamine (Et ₃ N), 50 ml of tetrahydrofurane (THF) is added and dissolved.
・Blow nitrogen gas through the above solution for 30 minutes to remove dissolved oxygen.
• Dissolve 9.3 g of 2-bromoisobutyryl bromide (BiBB) in 10 ml of THF and transfer it to a 200 ml flask.
・ While vigorously stirring the 2-bromoisobutyryl bromide solution in the flask on ice, add the above bis(2-hydroxyethyl)disulfide solution, stir on ice for 10 minutes, and then stir at room temperature for an additional 16 hours. .
・Add 200 ml of ultrapure water to the stirred solution, transfer it to a separating funnel, and extract with 50 ml of ethyl acetate three times.
・ Combine the above three ethyl acetate phases into one, wash with 50 ml of 1% NaHCO ₃ solution and 50 ml of ultrapure water, dehydrate the ethyl acetate phase with magnesium sulfate, remove magnesium sulfate by filtration, and use a rotary evaporator. Evaporate the solvent.
・Purify by silica gel column chromatography with dichloromethane as eluent.
- After distilling off the solvent with a rotary evaporator, dry overnight (about 16 hours) with a vacuum dry.

(3B) Coating of Surface Initiator on Gripping Portion of Nanotweezers 1 A gold film 301 is formed in advance on the gripping portions 130a and 130b.
・Weigh 4.52 mg of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide synthesized in (3A-2) above and add it to 1 ml of ethanol (EtOH) or N,N-dimethylformamide. (DMF) to prepare a 10 mM solution.
- The gripping portions 130a and 130b on which the gold film 301 is formed are immersed in the above 10 mM solution and allowed to stand at room temperature for 5 hours.
- After that, the gripping portions 130a and 130b are washed with ethanol and air-dried.
The S atom of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide bonds with the gold atom of the gold film 301, and bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide is gold. It is fixed to the surface of membrane 301 (see FIG. 14).

(3C) Synthesis of Zwitterionic Polymer Starting from Surface Initiator Here, synthesis of zwitterionic polymer using SBMA is described. The procedure is as follows.
・Add 2 ml of ultrapure water and 2 ml of methanol (MeOH) to dissolve 1.67 g of SBMA and 0.62 g of sodium bromide (NaBr).
・Dissolve 22.34 mg of copper (II) bromide (CuBr ₂ ) and 230.4 mg of Tris[2-(dimethylamino)ethyl]amine (Me ₆ TREN) in 5 ml of MeOH to prepare a 20 mM CuBr ₂ + 200 mM Me ₆ TREN stock solution. .
• Dissolve 0.44 g of L-ascorbic acid (L-AA) in 50 ml of ultrapure water to prepare a 50 mM stock solution.
• Add 750 μl of 20 mM CuBr ₂ +200 mM Me ₆ TREN stock solution to the above SBMA+NaBr solution, mix, then add 150 μl of 50 mM L-AA solution.
・While ultrasonicating the above solution with a bath sonicator, nitrogen gas is passed through for 5 minutes to remove dissolved oxygen.
・Immerse the supporting part of the nanotweezers coated with the surface initiator in the above solution from which dissolved oxygen has been removed, put it in a closed container, replace the air inside the container with nitrogen gas, and overnight at room temperature (about 16 hours) Leave still.
・Then, the nanotweezers supporting part is washed twice with a 1M NaBr solution and then with ultrapure water, and then air-dried.

As shown in FIG. 14, PSBMA is formed by radical polymerization of SBMA between Br and C of bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide as a surface initiator. The molecular length of PSBMA can be controlled by the amount of added monomer and the reaction time. As for MPC and CBMA, as in the case of SBMA, the surface initiator bis[2-(2'-bromoisobutyryloxy)ethyl]disulfide formed on the surface of the gripping portion is the starting point for the polymerization reaction. Polymerization can be performed by stacking monomers from the substrate surface.

The coating process on the gripping parts 130a and 130b is performed, for example, in the final step of the nanotweezers manufacturing process, and the coated nanotweezers can provide the user with nanotweezers corresponding to biological samples as a product.
Uncoated nanotweezers or nanotweezers coated with a surface initiator on a gold film are shipped as nanotweezers intermediate products, and the user performs the above-described coating treatment to produce nanotweezers compatible with biological samples. It can also be used as tweezers. For users who need coating treatment, a coating treatment kit, for example, equipment required for coating treatment including the coating agent described above, or a coating agent alone may be provided.

The effects of the nanotweezers 1 of the embodiment and example described above are summarized as follows.
(1) The nanotweezers 1 have a pair of openable/closable arms 13a and 13b, and electrostatic actuators 14a and 14b as driving units for opening and closing the pair of arms 13a and 13b. Each of the arms 13a and 13b is formed with a grasping portion 130a and 130b formed with a coating layer 330 having a zwitterionic structure. Coating 330 is a coating layer on the surface of the grip.
When the nanotweezers 1 configured in this way are immersed in a culture solution of cells or the like to be grasped, the surfaces of the grasping portions 130a and 130b show the double layer of the coating layer 330 as shown in FIG. 5(b). The portion of the ionic structure becomes a structure in which the water molecules 53 are hydrated by electrostatic interaction. Due to such action, the effect of suppressing non-specific adsorption and the effect of high water retention can be obtained. As a result, when a biological sample such as a cell is handled with the nanotweezers 1, non-specific adsorption of the biological sample to the gripping portions 130a and 130b and drying of the biological sample can be prevented. It is possible to provide nanotweezers with excellent biological sample handling properties.

(2) An example of the zwitterionic structure constituting the coating layer consists of molecules having positive and negative charges. It consists of a zwitterionic structure containing a zwitterionic polymer as shown in 9(c).
Another example of a zwitterionic structure that constitutes the coating layer is a zwitterionic structure that includes a positively charged molecule and a negatively charged molecule. For example, as shown in FIG. 9(b), it includes a plurality of positively charged molecules and negatively charged molecules. FIG. 9(d) shows a polymer having a zwitterionic structure containing positively charged molecules and negatively charged molecules.
A polymer having a zwitterionic structure, such as PMPC or PSBMA, can be used as the coating layer 330 . A polymer with a zwitterionic structure has a higher water retention capacity than the coating 330 with a zwitterionic structure shown in FIGS. 9(a) and 9(b). can be done.

(3) As shown in FIG. 3, a metal film, such as a gold film 301, is formed on the surfaces of the grips 130a and 130b, and contains a zwitterionic molecule having a thiol group (SH group) at its end, such as MPC-SH. A coating agent can be used to form a coating layer 330 on the surface of the gold film 301 .
That is, the metal film 3001 is formed between the base material surfaces of the gripping portions 130 a and 130 b of the nanotweezers and the coating layer 330 , and the coating layer 330 is formed containing atoms that bond with the metal film 301 . there is When a gold film is formed on the surfaces of the gripping portions 130a and 130b, the coating agent contains molecules with a zwitterionic structure having sulfur atoms at the ends that bond with the gold atoms of the gold film. can make you stronger.
As a result, the ends of the zwitterionic molecules can be reliably fixed to the surface of the gripping portion by the Au—S bond.

(4) The coating agent for forming the coating layer 330 of the nanotweezers 1 contains molecules having a zwitterion structure, and when a gold film is formed on the gripping portion base material surface, it binds to the gold film. selected from materials having thiol groups that

(5) The method for manufacturing the nanotweezers 1 of the embodiment includes a pair of arms 13a and 13b that can be freely opened and closed, each having grips 130a and 130b, and a pair of nanotweezers having a drive unit for opening and closing the pair of arms 13a and 13b. A tweezers intermediate is prepared, a metal film is formed on the surface of the base material of the gripping portions 130a and 130b of the nanotweezers intermediate, and a coating agent containing molecules having a zwitterionic structure is applied to the metal films of the gripping portions 130a and 130b. to form a coating layer 330 having a zwitterionic structure.

(6) When the gripping portions 130a and 130b are formed with a zwitterionic polymer as the coating layer 330, a nanotweezers intermediate is manufactured by coating the surfaces of the gripping portions with a surface initiator for polymerization reaction.
That is, the nanotweezer intermediate has a pair of openable and closable arms 13a and 13b having gripping portions 130a and 130b, and a driving portion for opening and closing the pair of arms 13a and 13b. A surface initiator for polymerizing a monomer having a zwitterionic structure is formed on the substrate surface.
On the surface of the grasping portion of the intermediate of such nanotweezers, a zwitterionic monomer is polymerized with a surface initiator as a starting point to form a zwitterionic polymer. Nanotweezers can be provided for gripping samples and the like.
In this way, an effective polymer brush can be easily formed on the handle surface. As a result, it is possible to obtain nanotweezers that are excellent in the effect of suppressing nonspecific adsorption and the effect of retaining water.

(7) A method for manufacturing a nanotweezers intermediate includes a pair of arms 13a and 13b that can be freely opened and closed having grip parts 130a and 130b, and a nanotweezers having a driving part for opening and closing the pair of arms 13a and 13b. A surface initiator for initiating polymerization of a monomer having a zwitterionic structure is prepared, and the surfaces of the substrates of the gripping portions 130a and 130b are coated with the surface initiator.
Another example of the nanotweezers intermediate has a pair of openable and closable arms 13a and 13b having gripping portions, and a driving portion for opening and closing the pair of arms 13a and 13b. A surface initiator is formed on the substrate surface to form a coating layer 330 having a zwitterionic structure.

(8) A method for producing nanotweezers in which the coating layer is composed of a zwitterionic polymer comprises preparing a nanotweezers intermediate in which a surface initiator is formed on the surface of the gripping portion, and producing a molecule having a zwitterionic structure. The coating agent containing the zwitterionic structure is applied to the gripping portions 130a and 130b to form a coating layer 330 having a zwitterionic structure starting from the surface initiator on the surfaces of the gripping portions 130a and 130b coated with the surface initiator.

(9) The nanotweezers kit includes the above coating agent and the above nanotweezers intermediate as a set. Nanotweezers with a coating layer that facilitates grasping and releasing biological samples such as cells are fabricated by users who handle biological samples themselves.

Although various embodiments and modifications have been described above, the present invention is not limited to these contents. Other aspects conceivable within the scope of the technical idea of the present invention are also included in the scope of the present invention.

Reference Signs List 1 nanotweezers 13a, 13b arm 14a, 14b electrostatic actuator 53 water molecule 55 zwitterion molecule 130a, 130b gripping portion 280 buffer solution 281 cell 300 coating agent, 301... gold film, 330... coating layer

Claims (10)

A pair of arms that can be opened and closed,
a driving unit for opening and closing the pair of arms,
Each of the arms is provided with a gripping portion having a coating layer of an oriented monomolecular film of molecules having a zwitterionic structure,
A metal film is formed on at least the surface of the base material of the grip part on which the coating layer is formed,
The coating layer is formed by the molecules having the zwitterionic structure containing atoms at their ends that bind to the metal of the metal film, and the atoms binding to the metal of the metal film, or Nanotweezers, which are formed by binding molecules having a ionic structure to the metal film via a surface initiator containing atoms that bind to the metal of the metal film. In the nanotweezers according to claim 1,
The nanotweezers, wherein the zwitterionic structure consists of molecules with positive and negative charges. In the nanotweezers according to claim 1,
The nanotweezers, wherein the coating layer is a zwitterionic structure containing positively charged molecules and negatively charged molecules. In the nanotweezers according to any one of claims 1 to 3,
the metal film is a gold film;
Nanotweezers, wherein said atoms are sulfur atoms. Preparing a nanotweezer intermediate having a pair of openable and closable arms having a gripping portion and a driving portion for opening and closing the pair of arms,
forming a metal film on at least the surface of the base material of the gripping portion of the nanotweezers intermediate on which the coating layer is formed;
applying a coating agent containing a molecule having a zwitterionic structure containing a sulfur atom that binds to the metal film to the surface of the metal film of the grip part;
A method for producing nanotweezers, comprising forming a coating layer of an oriented monomolecular film of molecules having the zwitterionic structure. A method of manufacturing the nanotweezers according to claim 5 ,
the metal film is a film made of gold or nickel;
The method for producing nanotweezers, wherein the coating agent contains a thiol group that binds to the metal film. A nanotweezers intermediate for producing the nanotweezers according to claim 1,
a pair of openable and closable arms having grips;
a driving unit for opening and closing the pair of arms,
A metal film is formed on the surface of the base material of the gripping part,
The nanotweezer intermediate, wherein the surface of the metal film is coated with a surface initiator for forming a coating layer of the oriented monolayer of molecules having the zwitterionic structure. A method for producing a nanotweezers intermediate for producing the nanotweezers according to claim 1,
preparing a nanotweezers having a pair of openable and closable arms having a gripping portion and a driving portion for opening and closing the pair of arms, and having a metal film formed on the substrate surface of the gripping portion;
preparing a surface initiator for initiating polymerization of a monomer having a zwitterionic structure and forming a coating layer of an oriented monomolecular film of molecules having the zwitterionic structure;
A method for producing a nanotweezers intermediate, wherein the surface of the metal film is coated with the surface initiator. preparing the nanotweezers intermediate according to claim 7 ;
applying a coating agent containing a molecule having a zwitterionic structure to the grip,
A method for producing nanotweezers, comprising the step of forming a coating layer of an oriented monomolecular film having the zwitterionic structure starting from the surface initiator on the surface of the gripping portion coated with the surface initiator. . A coating agent for forming the coating layer of the nanotweezers according to any one of claims 1 to 4, the coating agent comprising a molecule having a zwitterionic structure;
A pair of openable and closable arms having a gripping portion, and a driving portion for opening and closing the pair of arms, wherein a metal film is formed on a base material surface of the gripping portion,
A tweezers kit, comprising: a nanotweezers intermediate, wherein the surface of the metal film is coated with a surface initiator for forming a coating layer of the oriented monolayer having the zwitterionic structure.

Images