WO2017008699A1

WO2017008699A1 - Device for measuring cell traction force, and measuring method and preparation method

Info

Publication number: WO2017008699A1
Application number: PCT/CN2016/089401
Authority: WO
Inventors: 李舟; 金一鸣; 张亚岚; 郑强; 欧阳涵; 王中林
Original assignee: 北京纳米能源与系统研究所
Priority date: 2015-07-10
Filing date: 2016-07-08
Publication date: 2017-01-19
Also published as: CN106338500A; CN106338500B

Abstract

Disclosed are a device for measuring the cell traction force, and a measuring method and a preparation method, wherein the measuring device comprises: a substrate layer (3); a nanowire layer (1) located on the substrate layer (3), comprising nanowire arrays, wherein nanowires (11) can be bent under the effect of an external force; and a light-emitting layer (2), comprising luminous points (21) arranged on the end of each nanowire (11), and supporting the bending of the nanowire (11) of a supporting cell (4) to be tested, and driving the movement of the luminous point (21) on the end of nanowire (11) to produce the shifting displacement representing the cell (4) traction force after the cell (4) to be tested is arranged on the light-emitting layer (2). The device for measuring the cell traction force acquires the shifting displacement representing the cell (4) traction force in real time according to the movement situation of the luminous point (21) of the light-emitting layer (2), and has a high accuracy.

Description

Cell traction measuring device, measuring method and preparation method

Technical field

The present invention relates to the technical field of cell measurement, and in particular to a cell traction measuring device, a measuring method and a preparation method.

Background technique

Traction is one of the most important properties of cells. It is associated with many complex biological signal-mediated channels and plays an important role in cell proliferation, differentiation, contraction, migration and apoptosis. In addition, cell traction is highly correlated with the development and progression of many diseases, such as tumors. Therefore, some quantitative studies of individual cells are of great interest in both physiology and pathology.

Currently, cell traction is typically measured based on a soft nano/micro line array. For example, cell traction is calculated by measuring the amount of bending of the microcolumn based on a PDMS (polydimethylsiloxane) microcolumn array. However, this method has great limitations: (1) the measurement method is limited, and it is necessary to fix the cells and perform SEM (scanning electron microscope) observation, which cannot reflect the traction of living cells in real time; (2) soft A nanowire array (such as a PDMS microcolumn array) not only bends but also elastically deforms (axially stretches) when subjected to force, thereby affecting the accuracy of cell traction measurement; (3) nano-line shape according to SEM photographs It is subject to mechanical quantification, more interference by human factors, and greater error; (4) SEM price is more expensive, which is not conducive to popularization.

Summary of the invention

It is an object of the present invention to provide a measuring device and a measuring method for cell traction force, which can measure the traction force of a cell in real time.

In order to achieve the above object, the present invention provides a cell traction measuring device, the measuring device comprising: a base layer; a nanowire layer on the base layer, comprising a nanowire array, the nanowires in the array being used for And being capable of being bent by an external force; and an illuminating layer comprising a light-emitting point disposed at an end of each of the nanowires for supporting the cell to be tested after the cell to be tested is placed on the luminescent layer The nanowire is bent to drive the light-emitting point of the end of the nanowire to move, resulting in an offset displacement that characterizes the traction of the cell.

The measuring device for cell traction of the present invention can directly place the cell to be tested on the light emitting layer by providing a light emitting layer on the nanowire layer, and obtain the table in real time according to the movement of the light emitting point in the light emitting layer. The displacement displacement of the cell traction force is high and the accuracy is high.

Another object of the present invention is to provide a method for preparing a cell traction measuring device, the method comprising: providing a substrate layer; generating a nanowire layer on the substrate layer, the nanowire layer comprising a nanowire array The luminescent material is modified at the ends of the nanowires within the array to form a luminescent layer.

The preparation method of the cell traction measuring device of the invention can obtain the cell traction force measuring device by sequentially forming the nanowire layer and the luminescent layer on the substrate layer, and the preparation method is simple and convenient to operate.

Other features and advantages of the invention will be described in detail in the detailed description which follows.

DRAWINGS

The drawings are intended to provide a further understanding of the invention, and are intended to be a In the drawing:

1 is a schematic structural view of a measuring device for cell traction of the present invention;

2 is a front view showing a dynamic view of a cell traction measuring device of the present invention;

Fig. 3 is a top plan view showing the apparatus for measuring the cell traction force of the present invention.

Description of the reference numerals

1 nanowire layer 11 nanowire

2 luminescent layer 21 luminous point

3 basal layer 4 cells to be tested

detailed description

The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.

Directional terms such as "upper", "lower", "front", "back", "left", "right", etc., are used in the present invention, and are merely referring to the directions of the drawings. Therefore, the directional terminology used is intended to be illustrative of the scope of the invention.

As shown in FIG. 1 to FIG. 3, the measuring device for cell traction of the present invention comprises a base layer 3; a nanowire layer 1 on the base layer 3, comprising a nanowire array, the nanowires in the array being used for receiving an external force When functional, capable of bending; and the luminescent layer 2, including each of the nanowires 11 The light-emitting point 21 at the end of the nanowire 11 is used to move the light-emitting point 21 at the end of the nanowire 11 after the cell 4 to be tested is placed on the light-emitting layer 2 , producing an offset displacement that characterizes the cell's traction.

The measuring device for cell traction of the present invention can directly place the cell to be tested on the luminescent layer by disposing a luminescent layer on the nanowire layer, and obtain an offset displacement characterization of the cell traction force in real time according to the movement of the illuminating point in the luminescent layer. The accuracy is high; and in the measurement process, the cells can be detected without the need to fix the cells.

In order to reduce the influence of the elastic deformation of the nanowire on the measurement to improve the accuracy of the measurement, the nanowire 11 of the cell traction measuring device of the present invention is made of a hard material having a Young's modulus. Wherein, the hard material may be a metal, a non-metal or a compound thereof, for example, the metal may be germanium (Ge), lead (Pb), copper (Cu), silver (Ag) or platinum (Pt), etc. The non-metal may be silicon (Si) or selenium (Se), and the compound may be gallium nitride (GaN), silicon dioxide (SiO ₂ ), zinc oxide (ZnO) or zinc sulfide (ZnS), etc. Not limited to this.

Further, in order to facilitate observation and accurately obtain the offset displacement, the entire measuring device can be made transparent, and the nanowire 11 is made of a transparent material. For example, glass or quartz can be selected.

In order to increase the resolution of the detection, the density of the nanowires 11 in the nanowire layer 1 is 1 × 10 ⁵ /cm ² -1 × 10 ⁸ /cm ² . The nanowire 11 has an aspect ratio of 5-50; in general, the nanowire 11 has a length of 1-20 μm and a diameter of 50-500 nm.

The light-emitting point 21 in the light-emitting layer 2 is made of a light-emitting material. The luminescent material can be a quantum dot and/or a fluorescent dye. Wherein, the quantum dots may be cadmium sulfide (CdS), cadmium selenide (CdSe), cadmium telluride (CdTe), zinc sulfide (ZnS), etc.; the fluorescent dye may be fluorescein isothiocyanate , FITC), tetraethyl rhodamine B200 (RB200), tetraethyl rhodamine isothiocyanate (TRITC), phycoerythrin (R-Re), 4,6 - 4,6-diamidino-2-phenylindole (DAPI), etc., at a lower cost.

The invention also provides a method for measuring cellular traction. The measuring method of the cell traction force comprises: placing the cell to be tested on the luminescent layer in the measuring device for the cell traction force; and obtaining an offset displacement characterization of the cell traction force according to the movement of the illuminating point in the illuminating layer; The traction of the cells to be tested is determined.

During the measurement process, external stimuli, such as light, electricity, force or biochemical molecules, can also be applied to the cells to speed up the spreading of the cells and shorten the measurement time.

The invention does not need to use a more expensive SEM when measuring the cell traction force, and only needs to use an ordinary optical microscope (for example, a metallographic microscope or an inverted fluorescence microscope). Convenient and fast, low cost and wide range of use.

Further, the present invention provides a method of preparing a cell traction measuring device. The preparation method includes: providing a base layer; generating a nanowire layer on the base layer, the nanowire layer comprising a nanowire array; modifying a luminescent material at an end of the nanowire in the array to form a luminescent layer .

The method for generating a nanowire layer on the base layer includes: when the base layer and the nanowire layer are the same material, generally etching a nanowire layer on the substrate by etching; When the base layer and the nanowire layer are of different materials, a nanowire layer is grown and/or deposited on the base layer by techniques such as hydrothermal and/or epitaxial growth.

The etching method includes: at least one of Metal-Assisted Chemical Etching (MACE), Electron Beam Lithography (EBL), and Laser Interference Lithography (LIL). One.

A method of modifying a luminescent material at an end of a nanowire within the array includes: affixing a luminescent material to an end of the nanowire by a nano-seal technique; or coating a hydrophilic layer on the nanowire layer Etching the hydrophilic substance with a plasma or the like to expose an end of the nanowire; adding a luminescent material (the luminescent material is a hydrophobic substance), and modifying an end of the nanowire with the a luminescent material; the hydrophilic substance is dissolved using a solvent. The plasma may be an Inductively Coupled Plasma (ICP) or a Reactive Ion Etching (RIE), but is not limited thereto.

The method for preparing a cell traction measuring device of the present invention can obtain a cell traction force measuring device by sequentially forming a nanowire layer and a light emitting layer on a substrate layer, the preparation method is simple, the operation is convenient; and the prepared cell traction force measuring device structure Simple, high measurement accuracy and wide range of use.

The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the embodiments described above, and various modifications may be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variations are within the scope of the invention.

It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention has various possibilities. The combination method will not be described separately.

In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.

Claims

A measuring device for cell traction, characterized in that the measuring device comprises:

Base layer (3);

a nanowire layer (1) on the substrate layer (3), comprising a nanowire array, the nanowires in the array being capable of being bent when subjected to an external force;

a light-emitting layer (2) comprising a light-emitting point (21) disposed at an end of each of the nanowires (11) for supporting the cell (4) to be tested after being placed on the light-emitting layer (2) The nanowire (11) of the cell to be tested (4) is curved to drive the light-emitting point (21) of the end of the nanowire (11) to move, resulting in an offset displacement characterizing the traction force of the cell.
The cell traction measuring device according to claim 1, wherein the nanowire (11) is made of a hard material set to a Young's modulus.
The cell traction measuring device according to claim 2, characterized in that the nanowire (11) is made of a transparent material.
The cell traction measuring device according to any one of claims 1 to 3, wherein the nanowire (11) has an aspect ratio of 5 to 50.
The cell traction measuring device according to any one of claims 1 to 4, wherein the nanowire (11) has a length of 1 to 20 μm and a diameter of 50 to 500 nm.
The cell traction measuring device according to any one of claims 1 to 5, wherein the nanowire (11) in the nanowire layer (1) has a density of 1 × 10 5 /cm 2 -1 ×10 8 pieces/cm 2 .
The apparatus for measuring the traction force of a cell according to any one of claims 1 to 6, characterized in that the light-emitting point (21) is made of a luminescent material.
The apparatus for measuring the traction force of a cell according to claim 7, wherein said The luminescent material is a quantum dot and/or a fluorescent dye.
A method for measuring cell traction force, characterized in that the measurement method comprises:

The cell to be tested is placed on the luminescent layer in the cell traction measuring device according to any one of claims 1-8;

An offset displacement characterizing the cell traction force is obtained according to the movement of the light-emitting point in the light-emitting layer.
The method for measuring the traction force of a cell according to claim 9, wherein the measuring method further comprises:

The traction force of the cell to be tested is determined according to the offset displacement.
The method for measuring the traction force of a cell according to claim 9 or 10, wherein the measuring method further comprises:

Apply external stimuli to the cells to be tested.
A method for preparing a cell traction measuring device, characterized in that the preparation method comprises:

Providing a base layer;

Generating a nanowire layer on the substrate layer, the nanowire layer comprising a nanowire array;

A luminescent material is modified at the ends of the nanowires within the array to form a luminescent layer.
The method of preparing a cell traction measuring device according to claim 12, wherein the method of generating a nanowire layer on the base layer comprises:

When the base layer and the nanowire layer are the same material, the nanowire layer is etched on the substrate by etching;

When the substrate layer and the nanowire layer are of different materials, the nanowire layer is grown and/or deposited on the substrate layer by hydrothermal and/or epitaxial growth techniques.
The method for preparing a cell traction measuring device according to claim 12 or 13, wherein the method of modifying the luminescent material at the end of the nanowire in the array comprises:

Pasting a luminescent material at the end of the nanowire by a nano-seal technique; or

Coating a layer of hydrophilic substance on the nanowire layer; etching the hydrophilic substance by plasma to expose an end of the nanowire; adding a luminescent material to modify an end of the nanowire Place a luminescent material; the hydrophilic substance is dissolved using a solvent.