CN111077185B

CN111077185B - Multi-degree-of-freedom self-assembly nano robot and manufacturing control method thereof

Info

Publication number: CN111077185B
Application number: CN201911214317.4A
Authority: CN
Inventors: 司伟; 陈畅; 孙倩怡; 余梦; 沙菁; 章寅; 陈云飞
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2022-05-17
Anticipated expiration: 2039-12-02
Also published as: CN111077185A

Abstract

The invention relates to a multi-degree-of-freedom self-assembly nano robot and a manufacturing control method thereof. Depositing round gold electrodes on four vertexes of a square on a silicon-based material respectively, and processing four nano holes on the round gold electrodes; the size of the nanopore is such that each nanopore will only be able to capture one strand of deoxyribonucleic acid under the action of an applied electric field. The nano gold electrode is connected with an external voltage source, and the electrical property and the strength of the charge density on each nano hole can be regulated and controlled by regulating and controlling the direction and the magnitude of the voltage on each nano hole, so that the direction and the strength of the electroosmotic flow passing through the nano holes are controlled, combined or competitive driving is formed with the electric field force borne by the deoxyribonucleic acid chains, and the motion speed and the direction of the nano robot are controlled.

Description

Multi-degree-of-freedom self-assembly nano robot and manufacturing control method thereof

Technical Field

The invention relates to an electroosmosis and electrophoresis combined driving technology based on a nanopore; in particular to a multi-degree-of-freedom self-assembly nano robot and a manufacturing control method thereof.

Background

The optimal detection state of the single molecule detection technology based on the nano-pores is that molecules to be detected can be stably positioned in the nano-pores for a long time, and the molecules to be detected can be controlled to repeatedly enter and exit the nano-pores, so that repeated measurement is realized, and the detection error rate is reduced. Currently, the common single-molecule control technologies include Atomic Force Microscope (AFM), optical tweezers, magnetic tweezers, and the like. If the molecule to be detected and the AFM probe (or the micro-nano particles) are assembled and combined through a gold-thiol bond (or a strong interaction of biotin-streptavidin), the AFM probe inevitably damages the biomolecule to be detected and even enables the biomolecule to be detected to fall off from the surface of a needle tip when scanning the substrate of the nanopore, and in addition, due to the existence of mechanical and temperature drift, the time for stably capturing the molecule to be detected by the nanopore is short, and the success rate of experiments is low. Although the magnetic tweezers and the optical tweezers do not have the defect of mechanical drift, the nano holes are very small, so that the particles bound with the molecules to be detected are difficult to accurately move to the positions of the nano holes by combining an optical microscope. In addition, the AFM, magnetic tweezers and optical tweezers technologies are not suitable for being integrated with the nanopore chip, so that the portability of the nanopore-based detection chip is greatly reduced, and therefore if a nano robot exists, the nano robot can be integrated with the nanopore chip and can clamp molecules to be detected, the molecules to be detected are driven to move with sub-nanometer feeding precision, and the moving direction and speed are adjustable, so that the molecules to be detected are expected to be accurately sent into the nanopore, and the nano robot can be further controlled to capture the molecules to be detected by the nanopore again even if interference factors such as temperature drift exist.

The design concept of the micro-control robot proposed at present is mainly focused on the micrometer-millimeter scale, and is rarely reported after reaching the nanometer scale. In the human body, biomicromolecular machines such as bacterial flagellar motors and the enzyme F1F0 Adenosine Triphosphate (ATP) synthase are two typical rotating machines and play an extremely important role in the human body due to their unique structures and functions. If a nano molecular machine with similar functions can be designed and manufactured, the application prospect of the nano molecular machine is unimaginable and is certainly a great breakthrough in human history, and the nano molecular machine can benefit human beings in the aspects of disease diagnosis and treatment and drug design. With the development of DNA self-assembly technology in recent years, researchers have successfully established some nano-motion mechanisms (kinematic pairs) and nano-robots, etc. and realized certain motion operations, so that the design and manufacture of nano-molecular machines become possible. Professor lu Qian, california academy of Science, 2017, reported that they developed a nanocrobe assembled of single-stranded DNA that could autonomously walk on the surface of a substrate coated with DNA origami, seize certain molecules, and release them to a designated location, enabling the sorting of target cargo molecules. Based on this idea, if a nano-robot with a large range (nm- μm movable range) and capable of being combined with semiconductor chip technology can be designed and manufactured, molecules to be detected can be accurately delivered to the nano-holes, and then the related characteristics of the molecules to be detected can be researched by detecting the ion current of the nano-holes. In addition, the method has other wider application, such as drug site-specific delivery of minimally invasive surgery, site-specific excision of early tumor, and the like.

The molecule to be detected in the existing nanopore monomolecular sensor can not be stably, continuously and controllably placed at the nanopore, so that the sensor has the problem that enough effective characteristics of the monomolecular can not be detected.

Disclosure of Invention

The invention relates to a multi-degree-of-freedom self-assembly nano robot and a manufacturing control method thereof, and provides a method for effectively controlling molecules to be detected by adopting the self-assembly nano robot and realizing the driving of the nano robot under the condition of electroosmosis driving and electrophoresis driving combination or competitive driving, so that the accurate control of the molecules to be detected can be realized, and the research on the detailed characteristics of the single molecules to be detected is facilitated

The technical scheme is as follows:

the multi-degree-of-freedom self-assembly nano robot comprises a nano robot and a nano robot walking substrate; the nano robot walking substrate consists of a silicon substrate, four nano holes and four nano gold electrodes, wherein the four nano gold electrodes are respectively connected with four independent voltage sources; the nano robot consists of micro-nano particles and four deoxyribonucleic acid chains; the moving speed and the moving direction of the nano robot are controlled by regulating the direction and the strength of the independent voltage on the nano holes, so that the multi-degree-of-freedom movement of the nano robot is realized.

The invention further improves that: the core of the nano robot is micro-nano particles, four limbs of the nano robot are deoxyribonucleic acid chains, sulfydryl or biotin are modified at the tail ends of the four deoxyribonucleic acid chains, a gold film or streptavidin is wrapped on the surface of the micro-nano particles, and the deoxyribonucleic acid chains are bound on the micro-nano particles through strong interaction of the gold-sulfydryl or the streptavidin and the biotin.

The invention further improves that: the limb length of the nano robot can be adjusted by the synthesis length of the deoxyribonucleic acid chain.

The invention further improves that: and the nano robot is controlled by adopting a mode of electroosmotic flow driving and electrophoresis driving combined driving.

The control method for manufacturing the multi-degree-of-freedom self-assembly nano robot comprises the steps of preparing the nano robot and preparing a walking substrate of the nano robot;

wherein the preparation of the nano robot comprises the following steps:

step 1: firstly, the nano robot is synthesized and self-assembled, and the method comprises the following steps:

a. plating a layer of gold film on the surface of the micro-nano particles by adopting magnetron sputtering, and diluting the prepared micro-nano particles to 10 mu M in an ethanol solvent;

b. synthesizing a deoxyribose nucleic acid chain with a terminal containing a sulfhydryl-SH functional group by adopting a PCR amplification technology, and then diluting to prepare the required concentration of 10 mu M;

c. mixing the micro-nano particles plated with the gold film in the step a with the deoxyribonucleic acid solution prepared in the step b, and standing for 18 hours;

d. then 5 ml of 0.1% -0.2% Sodium Dodecyl Sulfate (SDS) and 0.1M sodium phosphate are added to the solution of step c; PH =7.4 and left standing at room temperature for 7 days;

e. the appropriate amount of 1M sodium chloride was added to the above solution 6-8 times with a time interval of about 4 hours between each addition.

Step 2: then taking out a trace solution from the solution containing the self-assembled nano robot in the step 1, characterizing by an Atomic Force Microscope (AFM), and if fewer than 4 deoxyribonucleic acid chains are bound on the micro-nano particles, not selecting the nano robot; if the number of the deoxyribonucleic acid chains bound on the micro-nano particles is equal to 4, the deoxyribonucleic acid chains can be directly taken out for later use by using an AFM probe; if more than 4 deoxyribonucleic acid chains are bound on the micro-nano particles, an AFM probe can be adopted to cut off the redundant deoxyribonucleic acid chains and then extract the redundant deoxyribonucleic acid chains for later use;

preparing a walking substrate of the nano robot; the method comprises the following steps:

A. processing the silicon-based film by adopting a micro-nano processing technology; firstly, processing a silicon substrate by gluing, exposing, developing and other operations through an etching technology, wherein the thickness of the silicon substrate is more than 10 nm;

B. a square area is selected on the silicon-based film, the length value of the square can be adjusted according to the length of four limbs of the nano robot, and the adjustable range is that the deoxyribonucleic acid chain cannot move out of the nanopore when the core of the nano robot moves in the rectangular range. Then four nanometer metal electrodes are deposited at four vertexes of the square, and then the nanometer metal electrodes are connected with an external voltage source;

C. respectively processing nano holes in the centers of the four nano metal electrodes;

D. finally, plating a silicon dioxide protective layer on the silicon-based film containing the nano metal electrode and the nano hole to prevent the nano gold electrode from being oxidized;

after the steps are completed, a silicon-based chip is placed between two liquid pools, the solution in the two liquid pools can only flow through the nano holes in the silicon-based chip, then the prepared nano robot is placed on one side of the silicon-based chip, the four deoxyribonucleic acid chains on the nano robot can be captured by the nano holes under the action of electric field force by adding external voltage to the two ends of the silicon-based chip, and the four deoxyribonucleic acid chains finally enter the nano holes at the four vertexes of the square of the silicon-based film respectively because the diameter of the nano holes only allows one deoxyribonucleic acid chain to enter.

The invention further improves that: wherein the step C is to adopt a Focused Ion Beam (FIB) or a Transmission Electron Microscope (TEM) to process a nanopore in the center of the nanogold electrode; the diameter of the nanopore is only processed to allow only a single deoxyribonucleic acid chain to pass through, but not to accommodate two or more deoxyribonucleic acid chains; when the independent voltage source is not started, the four deoxyribonucleic acid chains can respectively penetrate into the four nano holes in an electrophoresis mode under the driving of the electric field force.

The invention further improves that: and D, plating a layer of silicon dioxide film with the thickness of 2-3nm on the silicon substrate chip by adopting magnetron sputtering or atomic layer deposition equipment to prevent the gold electrode from being oxidized and the chip from being insulated, and simultaneously reducing the diameter of the nanopore to 2-3 nm.

The nano robot is formed by connecting micro-nano particles with gold (or streptavidin) spread on the surface and four deoxyribose nucleic acid chains with thiolated (or biotinylated) ends through gold-sulfhydryl bonds (or strong interaction of the streptavidin and biotin). The length of the deoxyribose nucleic acid chain containing sulfhydryl (or biotin) at the end can be obtained by PCR amplification reaction according to actual needs. Redundant deoxyribonucleic acid chains possibly connected can be cut off by an atomic force microscope probe, so that only four deoxyribonucleic acid chains are ensured on the nano robot.

Each deoxyribonucleic acid chain is mainly acted by the electric field force and the electrophoresis force in the nanometer hole. For a specific nanopore, when a deoxyribonucleic acid chain moves in an electrophoresis mode, when the direction of an introduced electroosmotic flow driving force is consistent with the direction of an electrophoresis driving force, the deoxyribonucleic acid chain moves towards the electrophoresis direction in an accelerated mode; when the direction of the electroosmotic flow driving force is opposite to the direction of the electrophoretic force and is larger than the electrophoretic force, the speed of the deoxyribonucleic acid chain via hole is gradually reduced, and the deoxyribonucleic acid chain via hole can even move along the reverse direction of the electrophoretic driving force. Therefore, the multi-degree-of-freedom self-assembly nano robot and the motion control method thereof can be used for stably controlling the motion speed and direction of the nano robot in real time and can realize accurate control on molecules to be detected bound at the core of the nano robot.

Has the advantages that:

the invention designs and synthesizes a nano robot formed by self-assembling micro-nano particles and four deoxyribonucleic acid chains, the robot walks on a silicon-based film containing nano holes positioned at four vertexes of a square and a nano gold electrode, the electroosmotic flow strength in the nano holes can be regulated and controlled by regulating the direction and the strength of a voltage source connected with the nano gold electrode, and the nano robot is driven under the condition of the combination or competitive driving of electroosmosis driving and electrophoresis driving, so that the accurate control of molecules to be detected can be realized, and the detailed characteristics of the single molecules to be detected can be conveniently researched. The beneficial effects are detailed as follows:

1. the nano robot has a large-range adjustable range. The effective range (10 nm-15 mu m) of the movement of the core of the nano robot can be controlled by synthesizing deoxyribose nucleic acid chains with sulfhydrylation (or biotinylation) at the tail ends and designing the side length of a square on a silicon substrate.

2. The nano robot has the functions of multi-degree-of-freedom movement, controllable movement speed and the like. The direction and the speed of electroosmotic flow in the nanometer hole can be directly changed by independently adjusting the direction and the strength of a voltage source connected with the nanometer gold electrode on the nanometer hole.

3. The nano robot can realize instant start and stop, high-precision feeding, compensation and the like. Under the condition that the voltage along the normal direction of the substrate film and the voltage source connected with the nanogold electrode are fully opened, the direction and the strength of the voltage and the voltage source are independently adjusted, so that the external force borne by the core of the nano robot under the combined driving of electrophoresis and electroosmosis is not zero (or zero), the start-stop and slow feeding of the nano robot can be realized, if the core of the nano robot deviates from a specified target position, the electrophoresis and electroosmosis strength and direction can be further adjusted to realize error compensation, and the core of the nano robot is stably positioned at a fixed point position.

4. The direction and the size of the voltage of four independent voltage sources connected with the nano-gold electrode can be independently adjusted to control the electrical property and the strength of the charge density on the nano-pores, so that electroosmotic flow with variable direction and strength is generated in the nano-pores, the electroosmotic flow driving and the electrophoresis driving are combined to independently adjust the resultant force and the direction of deoxyribonucleic acid chains in the four nano-pores, the speed and the direction of the nano-robot are controlled, and the multi-degree-of-freedom control of the self-assembly nano-robot is realized.

Drawings

Fig. 1 is a schematic view of the structure of the sensor according to the present invention.

In fig. 1: 1. the nano-particle-based DNA chip comprises a silicon-based film, 2. a nano-pore, 3. micro-nano particles, 4. a deoxyribonucleic acid chain, 5. a nano-gold electrode and 6, 7, 8 and 9-voltage sources.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings in conjunction with specific examples.

As shown in FIG. 1, the self-assembly nano robot of the present invention is composed of micro-nano particles 3 and four deoxyribonucleic acid chains 4, and the specific layout thereof is shown in FIG. 1. The silicon-based film 1 for the nanometer robot to walk is respectively provided with a nanometer hole 2 and a nanometer gold electrode 5 on the vertex of a square on the silicon-based film 1.

Sulfydryl or biotin is modified at the tail ends of the four deoxyribonucleic acid chains 4, a gold film or streptavidin is wrapped on the surface of the micro-nano particle 3, and the deoxyribonucleic acid chains 4 are bound on the micro-nano particle 3 through strong interaction of gold-sulfydryl bonds or streptavidin and biotin.

The invention relates to an early preparation process, a working process and a motion control method of a multi-degree-of-freedom self-assembly robot, which are as follows:

firstly, the nano robot shown in fig. 1 is subjected to synthetic self-assembly by the following method:

step 1:

a micro-nano processing technology is adopted to process the nano robot walking substrate shown in figure 1, and the specific steps are as follows:

a: firstly, processing a silicon substrate by gluing, exposing, developing and other operations through an etching technology, wherein the thickness of the silicon substrate is more than 10 nm;

b: selecting a square area on a silicon substrate, wherein the side length of the square area is selected according to the length of the thiol deoxyribose nucleic acid chain at the tail end synthesized in the step 1 as a reference, then processing a circular gold electrode with the thickness of 2nm and the diameter of 50nm at the top point of the square area, and manufacturing a lead for connecting with an external voltage source;

c: processing a 5nm nanometer hole in the center of the nanometer gold electrode by adopting a Focused Ion Beam (FIB) or a Transmission Electron Microscope (TEM);

d: a silicon dioxide film with the thickness of 2-3nm is plated on a silicon-based chip by adopting magnetron sputtering or atomic layer deposition equipment to prevent gold electrode oxidation and chip insulation, and the diameter of a nanopore is reduced to the thickness of 2-3 nm.

After the steps are completed, the silicon-based chip is placed between the two liquid pools, the solution in the two liquid pools can only flow through the nanopore on the silicon-based chip, then the nano robot prepared in the step 2 is placed at one side of the silicon-based chip, the external voltage is added at the two ends of the silicon-based chip, four deoxyribonucleic acid chains on the nano robot can be captured by the nanopore under the action of the electric field force, and the four deoxyribonucleic acid chains finally enter the nanopores at the four vertexes of the square of the silicon-based film respectively because only one deoxyribonucleic acid chain is allowed to enter the diameter of each nanopore.

The charge density and the electric property and the intensity of the charge density on the nano-pores can be regulated and controlled by independently regulating the independent voltage source connected with the nano-gold electrodes, so that the direction and the intensity of electroosmotic flow in the nano-pores are regulated, the resultant force borne by the nano-robot can be changed in real time under the condition of jointly driving with the electrophoretic force, and the nano-robot can move along the formulated path and direction. Through further binding the molecules to be detected on the micro-nano particles, the nano robot can realize accurate control of the molecules to be detected.

The above-mentioned embodiments further describe the objects, technical solutions and advantages of the present invention in detail.

It should be understood that the above-mentioned embodiments are only exemplary of the present invention, and are not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The multi-degree-of-freedom self-assembly nano robot is characterized in that: comprises a nano robot and a nano robot walking substrate; the nano robot walking substrate consists of a silicon substrate (1), four nano holes (2) and four nano gold electrodes (5), wherein the four nano gold electrodes (5) are respectively connected with four independent voltage sources (6, 7, 8 and 9); the nano robot consists of micro-nano particles (3) and four deoxyribonucleic acid chains (4); the moving speed and the moving direction of the nano robot are controlled by regulating the direction and the strength of the independent voltage on the nano hole (2), so that the multi-degree-of-freedom movement of the nano robot is realized; the core of the nano robot is micro-nano particles (3), four limbs of the nano robot are deoxyribonucleic acid chains (4), sulfydryl or biotin is modified at the tail ends of the four deoxyribonucleic acid chains (4), a gold film or streptavidin is wrapped on the surface of the micro-nano particles (3), and the deoxyribonucleic acid chains (4) are bound on the micro-nano particles (3) through strong interaction of gold-sulfydryl bonds or streptavidin and biotin; the limb length of the nano robot can be adjusted by regulating the synthesis length of the deoxyribonucleic acid chain (4); the nano robot is controlled by adopting a mode of electroosmotic flow driving and electrophoresis driving combined driving; the diameter of the nanopore is only processed to allow only a single deoxyribonucleic acid chain to pass through, but not to accommodate two or more deoxyribonucleic acid chains; when the independent voltage source is not started, the four deoxyribonucleic acid chains can respectively penetrate into the four nano holes in an electrophoresis mode under the driving of the electric field force.

2. The manufacturing control method of the multi-degree-of-freedom self-assembly nano robot is characterized by comprising the following steps of: the method comprises the steps of preparing a nano robot and preparing a walking substrate of the nano robot;

wherein the preparation of the nano robot comprises the following steps:

plating a layer of gold film on the surface of the micro-nano particles by adopting magnetron sputtering, and diluting the prepared micro-nano particles to 10 mu M in an ethanol solvent;

step b, synthesizing a deoxyribose nucleic acid chain with a sulfhydryl-SH functional group at the tail end by adopting a PCR amplification technology, and then diluting the deoxyribose nucleic acid chain to the required concentration of 10 mu M;

step c, mixing the micro-nano particles plated with the gold film in the step a with the deoxyribonucleic acid solution prepared in the step b, and standing for 18 hours;

step d, then adding 5 ml of 0.1-0.2% Sodium Dodecyl Sulfate (SDS) and 0.1M sodium phosphate into the solution in the step c; p H =7.4, and left to stand at room temperature for 7 days;

e, adding a proper amount of 1M sodium chloride into the solution for 6-8 times, wherein the time interval between each addition is about 4 hours;

step 2: then taking out a trace solution from the solution containing the self-assembled nano robot in the step 1, characterizing by an Atomic Force Microscope (AFM), and if fewer than 4 deoxyribonucleic acid chains are bound on the micro-nano particles, not selecting the nano robot; if the number of the deoxyribonucleic acid chains bound on the micro-nano particles is equal to 4, the deoxyribonucleic acid chains can be directly taken out for later use by using an AFM probe; if more than 4 deoxyribonucleic acid chains are bound on the micro-nano particles, an AFM probe can be adopted to cut redundant deoxyribonucleic acid chains and then extract the redundant deoxyribonucleic acid chains for later use;

step A, processing a silicon substrate by adopting a micro-nano processing technology; firstly, processing a silicon substrate through gluing, exposure and development operations and an etching technology, wherein the thickness of the silicon substrate is more than 10 nm;

b, selecting a square area on the silicon substrate, wherein the length value of the square area can be adjusted according to the length of four limbs of the nano robot, and the adjustable range is that the deoxyribonucleic acid chain cannot move out of the nanopore when the core of the nano robot moves in the square range;

then four nanometer metal electrodes are deposited at four vertexes of the square, and then the nanometer metal electrodes are connected with an external voltage source;

c, respectively processing nano holes in the centers of the four nano metal electrodes;

d, plating a silicon dioxide protective layer on the silicon substrate containing the nano metal electrode and the nano hole to prevent the nano gold electrode from being oxidized;

after the steps are completed, the silicon substrate is placed between the two liquid pools, the fact that the solutions in the two liquid pools can only circulate through the nano holes in the silicon substrate is guaranteed, then the prepared nano robot is placed on one side of the silicon substrate, the external voltage is added to the two ends of the silicon substrate, four deoxyribonucleic acid chains on the nano robot can be captured by the nano holes under the action of the electric field force, and the four deoxyribonucleic acid chains finally enter the nano holes in the four vertexes of the square of the silicon substrate respectively due to the fact that the diameter of the nano holes only allows one deoxyribonucleic acid chain to enter the nano holes.

3. The method for controlling the fabrication of the multi-degree-of-freedom self-assembled nano robot as claimed in claim 2, wherein: wherein the step C is to adopt a Focused Ion Beam (FIB) or a Transmission Electron Microscope (TEM) to process a nanopore in the center of the nanogold electrode; the diameter of the nanopore is only processed to allow only a single deoxyribonucleic acid chain to pass through, but not to accommodate two or more deoxyribonucleic acid chains; when the independent voltage source is not started, the four deoxyribonucleic acid chains can respectively penetrate into the four nano holes in an electrophoresis mode under the driving of the electric field force.

4. The method for controlling the fabrication of the multi-degree-of-freedom self-assembled nano robot as claimed in claim 2, wherein: and D, plating a silicon dioxide film with the thickness of 2-3nm on the silicon substrate by adopting magnetron sputtering or atomic layer deposition equipment to prevent the gold electrode from being oxidized and the chip from being insulated, and simultaneously reducing the diameter of the nanopore to 2-3 nm.