WO2017028340A1

WO2017028340A1 - Single cell control method based on light-induced dielectrophoresis technique

Info

Publication number: WO2017028340A1
Application number: PCT/CN2015/088944
Authority: WO
Inventors: 李志�; 张光烈; 李文荣
Original assignee: 深圳大学
Priority date: 2015-08-14
Filing date: 2015-09-06
Publication date: 2017-02-23
Also published as: CN105092679A; CN105092679B

Abstract

A single cell control method based on a light-induced dielectrophoresis technique, comprising the steps: A. fabricating a light-induced dielectrophoresis chip (20), wherein the light-induced dielectrophoresis chip (20) is composed of a three-layer structure: the lower layer being ITO glass coated with a hydrogenated amorphous silicon coating layer, the upper layer being ITO glass without a coating layer, and a micro-fluidic channel being encapsulated between the upper and lower ITO glass layers for injecting a solution required for operations; B. inputting a variable frequency alternating current signal to electrodes of the upper and lower ITO glass layers, and at the same time using incident light to irradiate the light-induced dielectrophoresis chip (20), so as to generate a non-uniform electric field in an irradiated area; and C. under the real-time observation of a microscope image system, realizing cell control by changing the frequency and amplitude of the alternating current signal. The control method has the advantages of low cost, simple operation and high efficiency.

Description

Single cell control method based on light-induced dielectrophoresis

Technical field

The invention relates to the field of single cell dynamics, in particular to a single cell control method based on light induced dielectrophoresis.

Background technique

Single cell control is an interdisciplinary frontier that analyzes the development of penetration between chemistry, biology, and medicine.

The existing single cell control technology mainly includes patch clamp combined with atomic force microscopy technology. Langer of the University of Tübingen in Germany took the lead in experimenting with patch clamp technology and atomic force microscopy (AFM) in 1997. In 2001, Zhang of New York University used a patch clamp/atomic force microscope system to study cell-specific membrane motion, membrane potential, and ion current measurement functions. In 2008, Pamir et al. of the University of Munich in Germany combined atomic force microscopy with planar patch clamp technique to study the relationship between external mechanical stimulation and membrane potential and ion channel current on lymphocytes.

How to accurately quantify the nano-scale mechanical stimulation of single cells and automatically detect the physiological information of cells at the same time has been widely concerned by researchers at home and abroad. Most studies stay in the simple test phase.

In the combination of planar patch clamps and atomic force microscopy, atomic force microscopy can only provide an external stimulus, no more uses, which makes the way to provide external mechanical stimulation is relatively simple. And its operation is very cumbersome, costly and time consuming.

Therefore, the prior art has yet to be improved and developed.

Summary of the invention

In view of the above deficiencies of the prior art, the object of the present invention is to provide a single cell control method based on light-induced dielectrophoresis technology, which aims to solve the problem that the existing single cell control method is very cumbersome, costly, time consuming and has no vision. Feedback questions.

The technical solution of the present invention is as follows:

A single cell control method based on light-induced dielectrophoresis technology, comprising the steps of:

A. A photo-induced dielectrophoresis chip is prepared. The photo-induced dielectrophoresis chip has a three-layer structure: a three-layer structure: the lower layer is ITO glass coated with a hydrogenated amorphous silicon coating, and the upper layer is ITO glass without coating. A microfluidic channel is encapsulated between the upper and lower layers of ITO glass for injecting a solution for the desired operation;

B. inputting an alternating frequency signal of a variable frequency to the electrodes of the upper and lower layers of the ITO glass, and simultaneously irradiating the light-inducing dielectrophoresis chip with the incident light to generate a non-uniform electric field in the irradiated region;

C. Under the real-time observation of the microscope image system, cell control is achieved by changing the frequency and size of the AC signal.

The single cell control method based on the light-induced dielectrophoresis technique, wherein the step of fabricating the light-induced dielectrophoresis chip in the step A specifically includes:

A1, cleaning the ITO glass substrate;

A2 depositing a hydrogenated amorphous silicon coating on the ITO glass substrate;

A3, coating a photoresist on the hydrogenated amorphous silicon coating;

A4, performing plate printing on the photoresist;

A5, contact corrosion to the ITO glass substrate;

A6, removing the photoresist;

A7. A conductive adhesive is applied to the area of the ITO glass substrate that is not covered with the hydrogenated amorphous silicon coating.

The single cell control method based on the light-induced dielectrophoresis technique, wherein the average dielectrophoretic force of the cells in a non-uniform electric field is described by the following formula:

Where F _DEP is the average dielectrophoretic force acting on the cell, R is the radius of the cell, ε _m is the dielectric constant of the solution in which the cell is located, E _rms is the root mean square value of the applied AC signal, and f _CM is Clausius-Mossotti Factor, the real part of the factor Re[f _CM ] is taken when calculating the average dielectrophoretic force.

The single cell control method based on light-induced dielectrophoresis technology, wherein the f _CM factor is defined as follows:

ε _p * and ε _m * are the complex dielectric constants of the cells and solutions, respectively.

The single cell control method based on photoinduced dielectrophoresis technology, wherein the complex permittivity is expressed as:

Where ε is the dielectric constant of the solution, σ is the conductivity, and ω is the frequency of the applied AC signal.

The single cell control method based on light-induced dielectrophoresis technology, wherein the cell rotation speed is:

Where E is the electric field strength, η is the viscosity of the solution, IM[f _CM ] is the imaginary part of the Clausius-Mossotti factor, and K is the coefficient.

Advantageous Effects: The present invention has the following advantages: First, the cost is low, and the light-induced dielectrophoresis platform used in the present invention is low in cost. Second, the operation is simple, the entire control process is basically automated, and only the cultured cells are placed in the container, and other processes are all completed by software. Third, the efficiency is high, and the present invention can perform a large number of cell operations in a short period of time due to the automation of the control process. Fourth, high-precision real-time operation, real-time manipulation of cells through visual feedback, improving the accuracy of operation.

DRAWINGS

1 is a flow chart of a preferred embodiment of a single cell control method based on photoinduced dielectrophoresis.

2 is a schematic view showing the structure of a light-induced dielectrophoresis platform in the present invention.

detailed description

The present invention provides a single cell control method based on a photoinduced dielectrophoresis technique, and the present invention will be further described in detail below in order to make the objects, technical solutions and effects of the present invention more clear and clear. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Please refer to FIG. 1. FIG. 1 is a flow chart of a preferred embodiment of a single cell control method based on a light-induced dielectrophoresis technique. As shown in the figure, the method includes the following steps:

S100, preparing a light-induced dielectrophoresis chip (ODEP chip), wherein the light-induced dielectrophoresis chip has a three-layer structure: the lower layer is ITO glass coated with a hydrogenated amorphous silicon coating, and the upper layer is uncoated (ie, does not contain hydrogenation) ITO glass coated with amorphous silicon, encapsulating a microfluidic channel between the upper and lower ITO glass for injecting the solution required for operation;

S200, inputting an alternating frequency signal of a variable frequency to the electrodes of the upper and lower ITO glass, and irradiating the light-inducing dielectrophoresis chip with the incident light to generate a non-uniform electric field in the irradiated region; first injecting the cell into the microfluidic channel And the medium (the medium is the solution to be operated, that is, the solution in which the cells are located). Then enter the AC signal.

S300, under the real-time observation of the microscope image system, by changing the frequency and size of the AC signal to achieve cell control.

Further, in the step S100, the step of fabricating the light-induced dielectrophoresis chip specifically includes:

S101, cleaning the ITO glass substrate;

Clean the surface of the ITO glass substrate to ensure the cleanliness of the contact surface.

S102, depositing a hydrogenated amorphous silicon coating (a-Si:H) on the ITO glass substrate;

A layer of hydrogenated amorphous silicon was deposited on the surface of the ITO glass substrate to a thickness of 1 micron.

S103, coating a photoresist on the hydrogenated amorphous silicon coating;

S104, performing a plate printing on the photoresist;

The stencil is to make a cover according to the specified pattern, the cover is placed on the surface of the photoresist, and the cover is irradiated with ultraviolet rays, and the uncovered photoresist is dissolved under the action of ultraviolet rays, and finally the photoresist having the same shape as the cover is obtained. Floor.

S105, contact etching to the ITO glass substrate; specifically, etching the surface of the photoinduced dielectrophoresis chip with oxalic acid to remove the hydrogenated amorphous silicon coating without covering the photoresist.

S106, removing the photoresist; removing the photoresist from the surface of the hydrogenated amorphous silicon coating.

S107. Applying a conductive adhesive to a region of the ITO glass substrate that is not covered with the hydrogenated amorphous silicon coating. That is, a conductive contact is added at a position where the surface of the ITO glass is not covered with the hydrogenated amorphous silicon coating.

After the upper ITO glass is cleaned, apply a conductive adhesive.

A microfluidic channel is encapsulated between the upper and lower layers of ITO glass, specifically a microfluidic channel is encapsulated by PDMS or double-sided tape.

In step S200, as shown in FIG. 2, a light-induced dielectrophoresis platform is first constructed. In addition to the ODEP chip 20 produced in step S100, the platform also requires an optical microscope 10, an optical projector (high resolution), a programmable signal generation circuit, and a host system to form a microscope image system. The host system includes: an image acquisition module, a microscopic vision algorithm processing module, a biochip drive controller, a virtual electrode generation module, and a display output module. The image acquisition module is configured to acquire an image of the optical microscope 10, and is processed by a microscopic vision algorithm processing module and displayed by a display output module, and the microscopic vision algorithm processing module also drives the biochip driver controller and The virtual electrode generation module signals to control the operation of both. The biochip drive controller is coupled to the programmable signal generation circuit to vary the signal frequency and magnitude. The programmable signal generating circuit connects the ODEP chip 20 through electrodes. The optical projector is disposed below the ODEP chip 20 for illuminating the incident light. The virtual electrode generating module is connected to the projector.

The optical microscope parameters are as follows:

Nikon CFI60 infinity optical system;

Electric focus, can move up and down (upper 13mm / 2mm);

Trinocular tube, light distribution: eyepiece / camera 100% / 0, 20% / 100%, 0/100%;

Eyepiece magnification: 10x;

Concentrator: waterproof, working distance: 7.2mm;

Objective lens: 20x, highly achromatic lens, nanocrystalline coating;

Stage: electric X-axis and Y-axis, resolution: 0.1 micron;

Ultraviolet cut filter block;

Fluorescence filter set: FITC/GFP.

After the platform is built, the biochip driver controller can send a signal to the programmable signal generation circuit, and then the programmable signal generation circuit inputs the variable frequency AC signal to the electrodes of the upper and lower layers of the ITO glass, and the optical projector utilizes the incident. Light illuminates the light-inducing dielectrophoresis chip to produce a non-uniform electric field in the illuminated area.

In the step S300, under the real-time observation of the microscope image system, by changing the frequency and size of the alternating current signal, the direction and size of the dielectrophoretic force received by the cell are changed to control the direction of cell movement, and high-speed real-time manipulation is realized. Nano entity.

The following focuses on how to control the direction of cell movement by changing the frequency and size of the AC signal.

The average dielectrophoretic force experienced by a cell in a non-uniform electric field can be described by the following formula:

Where F _DEP is the average dielectrophoretic force acting on the cell, R is the radius of the cell, ε _m is the dielectric constant of the solution in which the cell is located, E _rms is the root mean square value of the applied electric field (AC signal), f _CM is Clausius-Mossotti factor, the real part of the factor Re[f _CM ] is taken when calculating the average dielectrophoretic force, which is defined as follows:

ε _p * and ε _m * are the complex permittivity of the cell and the solution, respectively, and the complex permittivity (including ε _p * and ε _m *) in Equation 2 can be expressed as:

Where ε is the dielectric constant of the solution, σ is the conductivity, and ω is the frequency of the applied electric field (alternating current signal).

It can be seen that f _CM is a frequency dependent variable factor. Considering the alternating electric field with different frequencies, when the dielectrophoretic force and the electric field intensity change direction are the same, it is called positive dielectrophoresis; when the dielectrophoretic force and the electric field intensity change direction are opposite, it is called negative dielectrophoresis. Therefore, by changing the frequency of the applied electric field, the direction of the dielectrophoretic force to which the cells are subjected can be changed to achieve the purpose of controlling the direction of cell movement.

Since the biological cells are subjected to the polarization of the non-uniform electric field to generate the dipole moment, the torque generated by the dielectrophoretic force is balanced with the friction torque received in the medium, and the cell rotation speed is:

Where E is the electric field strength (AC signal strength), IM[f _CM ] is the imaginary part of the Clausius-Mossotti factor, K is the coefficient, and η is the viscosity of the solution. The dielectric properties of the cells can be estimated based on the relationship between the rotational speed of the cells and the dielectric constant of the cells.

The strength and direction of the dielectrophoretic force that a cell receives depends primarily on the dielectric properties of the medium and the cell, such as shape, size, and electric field frequency. The present invention utilizes light-induced dielectrophoretic force (ODEP) (when a certain frequency band is applied, a dominant force in electro-hydraulics) to identify and manipulate biological cells, and to separate nanoscale polymer particles. The ODEP chip is driven by a variable frequency AC signal, and the AC signal is input through the conductive contacts of the upper and lower layers of ITO glass. At this time, only a small portion of the solution layer is divided and a uniform electric field is generated in the solution layer. When incident light illuminates the ODEP chip, the optical conductivity of a-Si:H increases by several orders of magnitude due to the increase in the number of electron-hole pairs. Due to the reduced resistance of the incident light region, in the solution layer The partial pressure will be greatly increased, so that the a:Si:H in the incident light region will become an effective virtual electrode to generate a non-uniform electric field. This light-induced, non-uniform electric field produces a dielectrophoretic force, ie, light-induced dielectrophoretic force (ODEP), of the particles in the polarized region. Programmatic dynamic motion is achieved through optical microscopy and host systems, and automated capture, manipulation, separation and assembly of micro-nano entities are achieved without any manual interface. Therefore, the ODEP chip of the present invention can provide a method for efficiently realizing high-speed manipulation of micro-nano entities.

In summary, the present invention has the following advantages: First, the cost is low, and the light-induced dielectrophoresis platform used in the present invention is low in cost. Second, the operation is simple, the entire control process is basically automated, and only the cultured cells are placed in the container, and other processes are all completed by software. Third, the efficiency is high, and the present invention can complete the classification of a large number of cells in a short period of time due to the automation of the control process. Fourth, high-precision real-time operation, real-time manipulation of cells through visual feedback, improving the accuracy of operation.

The method of the invention solves the problem that the traditional dielectrophoresis chip requires complex and fine electrode processing, and dynamically generates different shapes of virtual electrodes through an optical projection device, thereby generating a non-uniform electric field, and the dielectrophoretic force acts on the micro-nano Particles, real-time manipulation of micro-nano particles, and real-time image output.

It is to be understood that the application of the present invention is not limited to the above-described examples, and those skilled in the art can make modifications and changes in accordance with the above description, all of which are within the scope of the appended claims.

Claims

A single cell control method based on light-induced dielectrophoresis, characterized in that it comprises the steps of:

A. A photo-induced dielectrophoresis chip is prepared. The photo-induced dielectrophoresis chip has a three-layer structure: a three-layer structure: the lower layer is ITO glass coated with a hydrogenated amorphous silicon coating, and the upper layer is ITO glass without coating. A microfluidic channel is encapsulated between the upper and lower layers of ITO glass for injecting a solution for the desired operation;

B. inputting an alternating frequency signal of a variable frequency to the electrodes of the upper and lower layers of the ITO glass, and simultaneously irradiating the light-inducing dielectrophoresis chip with the incident light to generate a non-uniform electric field in the irradiated region;

C. Under the real-time observation of the microscope image system, cell control is achieved by changing the frequency and size of the AC signal.
The single cell control method based on the light-induced dielectrophoresis technique according to claim 1, wherein the step of fabricating the light-induced dielectrophoresis chip in the step A comprises:

A1, cleaning the ITO glass substrate;

A2 depositing a hydrogenated amorphous silicon coating on the ITO glass substrate;

A3, coating a photoresist on the hydrogenated amorphous silicon coating;

A4, performing plate printing on the photoresist;

A5, contact corrosion to the ITO glass substrate;

A6, removing the photoresist;

A7. Conductive adhesive is applied to the area of the ITO glass substrate that is not covered with the hydrogenated amorphous silicon coating. mixture.
The single cell control method based on light-induced dielectrophoresis according to claim 1, wherein the average dielectrophoretic force of the cells in a non-uniform electric field is described by the following formula:

Where F DEP is the average dielectrophoretic force acting on the cell, R is the radius of the cell, ε m is the dielectric constant of the solution in which the cell is located, E rms is the root mean square value of the applied AC signal, and f CM is Clausius-Mossotti Factor, the real part of the factor Re[f CM ] is taken when calculating the average dielectrophoretic force.
The single cell control method based on light-induced dielectrophoresis according to claim 3, wherein the f CM factor is defined as follows:

ε p * and ε m * are the complex dielectric constants of the cells and solutions, respectively.
The single cell control method based on photoinduced dielectrophoresis according to claim 4, wherein the complex permittivity is expressed as:

Where ε is the dielectric constant of the solution, σ is the conductivity, and ω is the frequency of the applied AC signal.
The single cell control method based on light-induced dielectrophoresis according to claim 5, wherein the cell rotation speed is:

Where E is the electric field strength, η is the viscosity of the solution, IM[f CM ] is the imaginary part of the Clausius-Mossotti factor, and K is the coefficient.