CN111693444B - Spring nanowire detector for cell mechanics detection and detection method thereof - Google Patents
Spring nanowire detector for cell mechanics detection and detection method thereof Download PDFInfo
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Abstract
The invention discloses a spring nanowire detector for cell mechanics detection, which comprises a vertical three-dimensional nanowire spring or a horizontal wavy linear nanowire spring, wherein one end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is fixed on a micro-mechanical operation platform, and the tail end of the other end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is of a linear or hooked suspended structure; when the tail end of the other end of the straight three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is linear, a fluorescent marker is arranged on the surface of the tail end of the linear nanowire. The invention can test the relevant mechanical properties by controlling the real-time deformation observation of the nanowire spring, and the suspended nanometer probe is arranged at the end part, thereby realizing the nondestructive detection of cells.
Description
Technical Field
The invention relates to the field of cell mechanics tests, in particular to a spring nanowire detector for cell mechanics detection and a method for detecting cell mechanics.
Background
The detection of cell-related mechanical properties, including weight, force required for penetrating cell membranes, deformation of cell membranes and cytoskeletons, cell adhesion and the like, is of great significance for researching cell growth processes (such as adhesion, proliferation, differentiation and apoptosis) and detecting intracellular substances (ion channels, proteins and genetic substances), and is helpful for understanding the mechanism research of external force stimulation on changing cell states and controlling cell behaviors (such as drug delivery and gene regulation). Research shows that the parts of cells which generate mechanical action with the outside are mainly cell membrane structures and extracellular matrix structures. The cell membrane is an elastic membrane which is about 6 nanometers thick and is formed by a layer of phospholipid, a natural barrier is provided for the cell and the outside, therefore, mechanical puncture of the cell membrane based on a nanowire structure provides possibility for conveying of drugs and intracellular substance detection reagents, and the difficulty of operation is that large external force can penetrate the cell membrane without damaging the cell. In addition, the cells act with the substrate material through the extracellular matrix to form the action of force (such as adhesion force and traction force), thereby regulating the adhesion and migration of the cells. Therefore, how to accurately measure the external force required to penetrate a cell membrane with a thickness of nanometer and the force between the cell membrane and the culture substrate is the focus of the research on cell mechanics.
Currently, common methods for detecting cell mechanics are: culturing cells on the surface of a long vertical nanowire array, causing the deformation of the nanowire array in the horizontal direction due to the traction force of the cells and a substrate, and calculating the stress by measuring the deformation amount of the nanowire (Feng H et al, Nano Energy, 2018, 50: 504-); or culturing cells on the surface of a long vertical nanowire array, externally adding a flat plate for pressurizing, enabling the array to penetrate through a cell membrane, and testing the required stress; or measuring a force-displacement curve of the nanoneedle when the nanoneedle is inserted into or withdrawn from the cell based on a high-precision etching (e.g., focused ion beam) of the atomic force microscope tip, thereby obtaining a relevant parameter of cell mechanics (Obataya I,et al, Nano Lett. 2005, 5(1): 27-30， Aramesh M, et alnat Nanotechnol, 2019, 14: 791-. Obviously, the resolution of mechanical detection is limited by the simultaneous action of a large number of arrayed nanowires on cells, and the cells are damaged greatly by insertion into the cells. The preparation cost of the atomic force microscope tip test is high, and the shape design of the probe cannot be realized so as to meet different test requirements.
Therefore, there is a need to develop a cytomechanics detecting device that can simply, safely, accurately and individually design and realize the detection of the cytomechanics.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems and the defects in the prior art, the invention provides the spring nanowire detector which is good in mechanical property, wide in application range and low in manufacturing cost, and also relates to a preparation method and a detection method of the spring nanowire detector.
The technical scheme is as follows: a spring nanowire detector for cytomechanics detection, characterized in that: the micro-mechanical operation platform comprises a vertical three-dimensional nanowire spring or a horizontal wavy linear nanowire spring, wherein one end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is fixed on the micro-mechanical operation platform, and the tail end of the other end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is of a linear or hooked suspension structure; when the tail end of the other end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is linear, a fluorescent marker is arranged on the surface of the tail end of the linear nanowire.
The invention also relates to a method for detecting cell mechanics by the nanowire spring detector, which is characterized by comprising the following steps: the method comprises the following steps:
firstly, fixing a horizontal wavy linear suspended nanowire spring or a linear suspended three-dimensional nanowire spring with a hook-shaped structure at the tail end, and fixing the linear suspended three-dimensional nanowire spring or the horizontal wavy linear suspended nanowire spring with a fluorescent marker at the tail end on a micro-mechanical operation platform;
secondly, moving a micro-mechanical operating platform to enable the fluorescence-labeled nanowire spring cell mechanics detection device to be in contact with cells, gradually reducing the height of the platform, observing the change of fluorescence intensity at the end part of the nanowire through a laser confocal fluorescence microscope to judge whether the detection device penetrates through a cell membrane to enter the cell, and detecting the force required by the penetration of the cell membrane through the relationship between the spring deformation and the mechanics;
or hooking the cells by using the nanowire spring with the hook-shaped end structure, applying tension to separate the cells from the surface of the substrate, and detecting the adhesion of the cells on the surface of the substrate through the relationship between the deformation of the spring and the mechanics;
or treating the spread cells by pancreatin, hooking the cells by the hook-shaped nanowire spring after the cells begin to shrink, separating the cells from the substrate, and detecting the weight of the cells through the relationship between the deformation of the spring and the mechanics.
The invention further defines the technical scheme as follows: the fluorescent marker is a pH value fluorescent probe, a calcium ion fluorescent probe, a hypochlorite fluorescent probe or an active oxygen fluorescent probe.
Preferably, in the second step, the cells comprise stem cells, nerve cells, cardiac muscle cells, cartilage cells, osteoblasts, fibroblasts or tumor cells.
Preferably, in the first step, the diameter range of the nanowires of the horizontal wavy linear suspended nanowire spring and the linear suspended three-dimensional nanowire spring is 20-200 nanometers, the inner diameter of the spring is 1-15 micrometers, and the number of the coils of the spring is 2-10 coils.
Preferably, the preparation method of the horizontal wavy linear suspended nanowire spring and the linear suspended three-dimensional nanowire spring in the first step comprises the following steps:
s1, on the silicon oxide wafer substrate, obtaining a columnar guide step through Bosh process etching assisted by polystyrene spheres or photoetching a wavy guide step on the surface of a plane substrate;
s2, placing the guide step in a plasma enhanced chemical vapor deposition system, taking indium with a low melting point as a catalyst, and growing a vertical three-dimensional nanowire spring or a horizontal wavy linear nanowire spring at a low temperature through a planar solid-liquid solid growth mechanism;
s3, cutting the tail end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring obtained in the second step by adopting a photoetching technology and an etching technology to obtain the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring with the linear or hooked end part, transferring the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring to a new substrate, and suspending and fixing the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring;
s4, modifying the end surfaces of the obtained vertical three-dimensional nanowire springs with linear ends and the horizontal wavy linear nanowire spring structures with fluorescent markers.
Preferably, in the first step, the linear suspended nanowire spring is prepared by optical fiber take-up transfer, supercritical drying after hydrofluoric acid treatment or dry etching after PMMA assisted transfer.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the nanowire spring is made of traditional semiconductor silicon materials, has good biocompatibility and excellent mechanical properties, can be used for testing relevant mechanical properties by controlling the real-time deformation observation of the nanowire spring, and can realize the nondestructive detection of cells by arranging the suspended nanoscale probe at the end part.
2. The detection method for cell mechanics provided by the invention is based on mechanical force to puncture cell membranes, and the mechanical measurement of the cell membranes is not limited by cell types and has a wide application range.
3. Compared with a metal wire, the metal wire has more excellent diameter and appearance controllability, can be used for preparing mechanical detection devices with different accuracies and application scenes, and has the accuracy controlled by the diameter and the inner diameter of the spring, wherein the smaller the diameter and the inner diameter is, the higher the accuracy is. The silicon nanowire prepared by the traditional photoetching process has low price and is easy to realize batch production by depending on mature preparation processes of semiconductor industry, suspended nanometer devices, plane-guided self-assembly growth and the like.
Drawings
FIG. 1 is a flow chart of the preparation of silicon nanowire springs in examples 1-4 of the present invention;
FIG. 2 is a schematic diagram of the external force test applied to the cell membrane in example 1 of the present invention;
FIG. 3 is a diagram illustrating the external force test applied to the cell membrane in example 2 of the present invention;
FIG. 4 is a schematic diagram of the cell gravity test in example 3 of the present invention;
FIG. 5 is a schematic diagram of the cell membrane adhesion test in example 4 of the present invention.
Detailed Description
The invention is further elucidated with reference to the drawings and the embodiments.
Example 1
The present embodiment provides a nanowire spring detector for detecting cell membrane penetrating force and a method for manufacturing the same, as shown in fig. 1-2, the method includes the following steps:
A. cleaning a silicon oxide wafer substrate material, obtaining a columnar guide step by etching with a Bosh process assisted by polystyrene spheres, then placing the columnar guide step in a plasma enhanced chemical vapor deposition system, taking indium with a low melting point as a catalyst, and growing a vertical three-dimensional nanowire spring of a nanowire at a low temperature by a planar solid-liquid-solid (IPSLS) growth mechanism. The diameter of the nanowire in this example is 50 nanometers, the inner diameter of the spring is 1 micrometer, and the number of turns of the spring is 4.
B. And C, cutting off the tail end of the vertical three-dimensional nanowire spring on the substrate obtained in the step A through a photoetching technology and an etching technology to obtain a nanowire structure with a linear end part, and transferring the nanowire structure to a new substrate to be suspended and fixed.
C. And D, performing surface pH value fluorescent probe modification on the nanowire with the linear end part obtained in the step B to obtain the pH value fluorescence labeled nanowire spring.
D. Fixing the nanowire spring cell mechanics detection device with the pH value obtained in the step C and marked by fluorescence on a micro-mechanical operation platform, gradually reducing the height of the platform, observing the change of the fluorescence intensity of the nanowire spring through a laser confocal fluorescence microscope, judging whether the detection device penetrates through a cell membrane, and detecting the force required by penetrating through the cell membrane through a spring force-displacement curve.
Preferably, the fluorescent label in this embodiment includes, but is not limited to, a pH fluorescent probe, a calcium ion fluorescent probe, a hypochlorite fluorescent probe, or an active oxygen fluorescent probe.
Preferably, in the step B, the suspended nanowire spring is prepared by methods including, but not limited to, optical fiber picking transfer, supercritical drying after hydrofluoric acid treatment, or dry etching after PMMA assisted transfer.
Preferably, the cells detected in this embodiment include, but are not limited to, stem cells, nerve cells, cardiac muscle cells, cartilage cells, osteoblasts, fibroblasts, or tumor cells.
Example 2
The present embodiment provides a method for detecting the amount of force required to penetrate a cell membrane by using a calcium ion fluorescence-labeled nanowire spring, as shown in fig. 1 and 3, the method includes the following specific steps:
A. cleaning a silicon oxide wafer substrate material, photoetching and etching a wave-shaped guide step on the surface of a plane substrate, then placing the silicon oxide wafer substrate material in a plasma enhanced chemical vapor deposition system, and growing a horizontal nanowire wave-shaped linear spring at a low temperature by using a plane solid-liquid-solid (IPSLS) growth mechanism with indium with a low melting point as a catalyst. The diameter of the nanowire is 80 nanometers, the inner diameter of the spring is 5 micrometers, and the number of turns of the spring is 5 turns;
B. and C, cutting off the tail end of the horizontal nanowire wavy linear spring on the substrate obtained in the step A through a photoetching technology and an etching technology to obtain a nanowire structure with a linear end part, and transferring the nanowire structure to a new substrate to be suspended and fixed.
C. And D, performing surface calcium ion fluorescent probe modification on the nanowire with the linear end part obtained in the step B to obtain the nanowire spring marked by calcium ion fluorescence.
D. Fixing the nanowire spring cell mechanics detection device obtained in the step C on a micro-mechanical operation platform, gradually reducing the height of the platform, observing the change of the fluorescence intensity of the nanowire spring through a laser confocal fluorescence microscope, judging whether the detection device penetrates through a cell membrane, and detecting the force required by penetrating through the cell membrane through a spring force-displacement curve.
Example 3
The embodiment provides a method for detecting the weight of a cell by using a hook-shaped nanowire spring, which comprises the following specific steps as shown in fig. 1 and 4:
A. cleaning a silicon oxide wafer substrate material, photoetching and etching a wave-shaped guide step on the surface of a plane substrate, then placing the silicon oxide wafer substrate material in a plasma enhanced chemical vapor deposition system, and growing a horizontal nanowire wave-shaped linear spring at a low temperature by using a plane solid-liquid-solid (IPSLS) growth mechanism with indium with a low melting point as a catalyst. The diameter of the nanowire is 60 nanometers, the inner diameter of the spring is 3 micrometers, and the number of turns of the spring is 5.
B. And C, cutting off the tail end of the horizontal nanowire wavy linear spring on the substrate obtained in the step A by using a photoetching technology and an etching technology to obtain a nanowire structure with a hook-shaped end part, and transferring the nanowire structure to a new substrate to be suspended and fixed.
C. And B, fixing the hook-shaped nanowire spring cell mechanical detection device obtained in the step B on a micro-mechanical operation platform, moving the position of the nanowire spring when the pancreatin-treated cells start to shrink balls, enabling the nanowire spring to be in contact with stem cells and hook the cells, upwards pulling the cells to enable the cells to be separated from the surface of the substrate, and detecting the weight of the cells through a spring force-displacement curve.
Example 4
The present embodiment provides a method for detecting cell adhesion by using a hook-shaped nanowire spring, as shown in fig. 1 and 5, the method includes the steps of:
A. cleaning a silicon oxide wafer substrate material, photoetching and etching a wave-shaped guide step on the surface of a plane substrate, then placing the silicon oxide wafer substrate material in a plasma enhanced chemical vapor deposition system, and growing a horizontal nanowire wave-shaped linear spring at a low temperature by using a plane solid-liquid-solid (IPSLS) growth mechanism with indium with a low melting point as a catalyst. The diameter of the nanowire is 60 nanometers, the inner diameter of the spring is 3 micrometers, and the number of turns of the spring is 5;
B. cutting off the tail end of the horizontal nanowire wavy linear spring on the substrate obtained in the step A by using a photoetching technology and an etching technology to obtain a nanowire structure with a hook-shaped end part, and transferring the nanowire structure to a new substrate to be suspended and fixed;
C. and B, fixing the hook-shaped nanowire spring cytomechanics detection device obtained in the step B on a micro-mechanical operation platform, moving the position of the nanowire spring cytomechanics detection device to enable the nanowire spring cytomechanics detection device to be in contact with osteoblasts and hook the osteoblasts, then upwards pulling up the detection device to enable the cells to be separated from the surface of the substrate, and detecting the adhesion force of the cells on the surface of the substrate through a spring force-displacement curve.
The embodiments of the present invention listed as preferred examples 1-4 are to detect cell-related mechanical properties by the deformation of the highly sensitive silicon nanowire spring structure when it is in contact with a cell. The method comprises the steps of respectively preparing three-dimensional and two-dimensional plane silicon nanowire spring structures on a cylinder and a plane by utilizing a programmable self-assembly guiding method, obtaining a linear or hook-shaped end part by an etching technology, then carrying out surface fluorescent substance modification on the silicon nanowire with the linear end part, monitoring the contact state of the nanowire structure and a cell in real time by combining a fluorescence microscope, recording the contraction deformation of the nanowire when the nanowire enters the cell, calculating the stress required by passing through a cell membrane, or realizing the detection of the weight of the cell and the bonding force between the nanowire spring structure and a substrate by utilizing the hook-shaped end part. The invention can realize high-sensitivity detection on cell mechanics, and simple detection equipment can be used for detecting cells at any angle; the nanoscale probe can realize nondestructive cell detection, and the nanowire spring structure can be produced in batches and is low in cost by relying on a mature self-assembly preparation technology.
The foregoing is only a preferred embodiment of this invention and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the invention and these modifications should also be considered as the protection scope of the invention.
Claims (7)
1. A spring nanowire detector for cytomechanics detection, characterized in that: the micro-mechanical operation platform comprises a vertical three-dimensional nanowire spring or a horizontal wavy linear nanowire spring, wherein one end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is fixed on the micro-mechanical operation platform, and the tail end of the other end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is of a linear or hooked suspension structure;
when the tail end of the other end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring is linear, a fluorescent marker is arranged on the surface of the tail end of the linear nanowire.
2. A method for detecting cell mechanics by a nanowire spring detector is characterized by comprising the following steps: the method comprises the following steps:
firstly, fixing a horizontal wavy linear suspended nanowire spring or a linear suspended three-dimensional nanowire spring with a hook-shaped structure at the tail end, and fixing the linear suspended three-dimensional nanowire spring or the horizontal wavy linear suspended nanowire spring with a fluorescent marker at the tail end on a micro-mechanical operation platform;
secondly, moving a micro-mechanical operating platform to enable the fluorescence-labeled nanowire spring cell mechanics detection device to be in contact with cells, gradually reducing the height of the platform, observing the change of fluorescence intensity at the end part of the nanowire through a laser confocal fluorescence microscope to judge whether the detection device penetrates through a cell membrane to enter the cell, and detecting the force required by the penetration of the cell membrane through the relationship between the spring deformation and the mechanics;
or hooking the cells by using the nanowire spring with the hook-shaped end structure, applying tension to separate the cells from the surface of the substrate, and detecting the adhesion of the cells on the surface of the substrate through the relationship between the deformation of the spring and the mechanics;
or treating the spread cells by pancreatin, hooking the cells by the hook-shaped nanowire spring after the cells begin to shrink, separating the cells from the substrate, and detecting the weight of the cells through the relationship between the deformation of the spring and the mechanics.
3. The method for detecting cell mechanics of a nanowire spring probe according to claim 2, wherein: the fluorescent marker is a pH value fluorescent probe, a calcium ion fluorescent probe, a hypochlorite fluorescent probe or an active oxygen fluorescent probe.
4. The method for detecting cell mechanics of nanowire spring probe of claim 3, wherein: in the second step, the cells comprise stem cells, neural cells, cardiac muscle cells, chondrocytes, osteoblasts, fibroblasts or tumor cells.
5. The method for detecting cell mechanics of nanowire spring probe of claim 4, wherein: in the first step, the diameter range of the nanowires of the horizontal wavy linear suspended nanowire spring and the linear suspended three-dimensional nanowire spring is 20-200 nanometers, the inner diameter of the spring is 1-15 micrometers, and the number of the coils of the spring is 2-10 coils.
6. The method for detecting cell mechanics of a nanowire spring probe according to claim 2, wherein: the first step is that the preparation method of the horizontal wavy linear suspended nanowire spring and the linear suspended three-dimensional nanowire spring comprises the following steps:
s1, on the silicon oxide wafer substrate, obtaining a columnar guide step through Bosh process etching assisted by polystyrene spheres or photoetching a wavy guide step on the surface of a plane substrate;
s2, placing the guide step in a plasma enhanced chemical vapor deposition system, taking indium with a low melting point as a catalyst, and growing a vertical three-dimensional nanowire spring or a horizontal wavy linear nanowire spring at a low temperature through a planar solid-liquid solid growth mechanism;
s3, cutting the tail end of the vertical three-dimensional nanowire spring or the horizontal wavy linear nanowire spring obtained in the second step by adopting a photoetching technology and an etching technology to obtain the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring with the linear or hooked end part, transferring the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring to a new substrate, and suspending and fixing the horizontal wavy linear nanowire spring or the vertical three-dimensional nanowire spring;
s4, modifying the end surfaces of the obtained vertical three-dimensional nanowire springs with linear ends and the horizontal wavy linear nanowire spring structures with fluorescent markers.
7. The method for detecting cell mechanics of nanowire spring probe of claim 6, wherein: in the first step, the preparation method of the linear suspended nanowire spring is optical fiber thread picking transfer, supercritical drying after hydrofluoric acid treatment or dry etching after PMMA assisted transfer.
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