WO2017028342A1

WO2017028342A1 - Cell classification method based on light-induced dielectrophoresis technique

Info

Publication number: WO2017028342A1
Application number: PCT/CN2015/088946
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: CN105044192B; CN105044192A

Abstract

A cell classification method based on a light-induced dielectrophoresis technique, comprising: fabricating a light-induced dielectrophoresis chip composed of a three-layer structure; inputting a variable frequency alternating current signal to electrodes of upper and lower ITO glass layers of the light-induced dielectrophoresis chip, and using incident light to irradiate the light-induced dielectrophoresis chip; changing the frequency and magnitude of the alternating current signal so as to control a cell motion direction, and at the same time collecting an image of a cell; and performing fitting, according to the collected image, on the displacement of a cell to be classified and a time, and using, according to a fitting result, a trained support vector machine for classification so as to obtain a cell type. The classification method is low in cost, simple in operation and high in accuracy, being able to reach 97.5 percent of accuracy.

Description

Cell classification method based on light-induced dielectrophoresis

Technical field

The invention relates to the field of cell classification, in particular to a cell classification method based on light-induced dielectrophoresis.

Background technique

In the prior art, cell classification is mainly the following methods:

1. Centrifugation: Centrifugation is a technique for classifying different cells from tissue homogenate or blood by using different sizes and densities of cells to settle in a centrifugal field. The main methods relying on centrifugation to separate cells are differential centrifugation, rate-zone density gradient centrifugation, iso-density centrifugation, and cell flotation centrifugation using a special rotor.

2, membrane filtration method: semi-permeable membrane as a selection barrier, the use of membrane selectivity (pore size), the energy difference between the two sides of the membrane as a driving force, allowing certain types of cells to pass through while retaining other Type cells to achieve separation of cell sorting techniques.

3. Fluorescence-activated cell sorting: the cells are bound by fluorescein-labeled antibody, and a single-flow cell is excited by a laser beam, and the cells are automatically analyzed or sorted according to the fluorescence carried by the cell. The principle is that fluorescently stained cells produce scattered light and excited fluorescence under the illumination of a laser beam. The light scattering signal is detected at a small forward angle. This signal basically reflects the volume of the cell; the direction of the fluorescence signal is perpendicular to the laser beam, and is separated by a series of dichroic mirrors and bandpass filters. Multiple different wavelengths Fluorescent signal.

However, the above methods have their shortcomings:

Centrifugal method: the cost is too high, it takes too long, and it is necessary to prepare a medium solution, which is strict in operation and difficult to control.

Membrane filtration method: The preparation of the membrane is too complicated. Different membranes have different requirements for the experimental environment, and the membrane has a limited service life.

Fluorescence-activated cell sorting: the cost is too high, and there are also low-abundance molecules in the cell that are difficult to detect, lack of "universal" cell-permeable substances, interference effects of cell autofluorescence, overlap of fluorescence intermolecular emission spectra, and lack of identifiable targets Molecular reagents. Especially for cell sorting, there are cell survival problems due to droplet formation, sorting cell re-analysis or pre-culture dilution, and the time delay required to obtain a sufficient number of available cells. Finally, data analysis is difficult to deal with, especially when dealing with low-abundance objects.

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 cell classification method based on light-induced dielectrophoresis technology, which aims to solve the problems of high cost, complicated operation, low efficiency and low accuracy of the existing cell classification method.

The technical solution of the present invention is as follows:

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

A. Making a light-induced dielectrophoresis chip, the light-induced dielectrophoresis chip having a three-layer structure Composition: the lower layer is ITO glass coated with hydrogenated amorphous silicon coating, the upper layer is ITO glass without coating, and a microfluidic channel is encapsulated between the upper and lower ITO glass for injecting the solution required for 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. Change the frequency and size of the AC signal to control the direction of cell movement while collecting images of the cells;

D. According to the acquired image, the displacement and time of the cells to be classified are fitted, and then the trained support vector machine is used to classify according to the fitting result, and the cell type is obtained.

The cell classification 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 cell sorting 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 cell classification 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 cell classification 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 cell classification method based on the light-induced dielectrophoresis technique, 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.

The cell classification method based on the light-induced dielectrophoresis technique, wherein the displacement formula of the cells under the average dielectrophoretic force is as follows:

U ₀ is the initial velocity of the cell under the average dielectrophoretic force, and τ is the time constant.

Advantageous Effects: The cell classification method of the present invention has low cost and simple operation, and the entire classification process is basically automated, and the cultured cells can be automatically classified by simply placing the cultured cells into the container, and the classification process is automated, so A large number of cells can be sorted in a short period of time with high efficiency. In addition, the method of the present invention has high accuracy and can achieve an accuracy of 97.5% under the existing conditions.

DRAWINGS

1 is a flow chart of a preferred embodiment of a cell sorting 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 cell sorting 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 cell classification method based on a light-induced dielectrophoresis technique. As shown in the figure, the method includes the following steps:

S100, manufacturing a light-induced dielectrophoresis chip (ODEP chip), wherein the light-induced dielectrophoresis chip has a three-layer structure: the lower layer is an ITO glass coated with a hydrogenated amorphous silicon coating, The layer is an uncoated ITO glass (ie, does not contain a hydrogenated amorphous silicon coating), and a microfluidic channel is encapsulated between the upper and lower ITO glass for injecting a solution for the desired 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, changing the frequency and size of the alternating signal to control the direction of cell movement while collecting images of the cells;

S400. Fit the displacement and time of the cells to be classified according to the acquired image, and then perform classification according to the fitting result using the trained support vector machine to obtain a cell type.

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 corrosion to the ITO glass matrix; specifically the core made of oxalic acid corrosion The surface layer is removed 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 (100 micron high) is packaged 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. 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 collect an image of the optical microscope 20, and is processed by a microscopic vision algorithm processing module and displayed by a display output module, wherein the microscopic vision algorithm processing module further 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 frequency and magnitude of the AC signal. 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 coupled to the optical 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, 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 the image of the cell is collected to realize high-speed manipulation of the micro-nano entity.

The following describes 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, IM[f _CM ] is the imaginary part of the Clausius-Mossotti factor, K is the coefficient, and η is the viscosity of the medium (ie, the solution in which the cells are located). 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 the cells are subjected to depends mainly on the dielectric of the medium and the cells. Characteristics 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. Since the resistance of the incident light region is reduced, the partial pressure in the solution layer is greatly increased, so that 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 realize high-speed manipulation of micro-nano entities.

Dielectrophoretic forces result from non-uniform electric fields, whereas conventional methods require complex manufacturing processes and preparation processes, and often limit cell movement. The optically induced dielectrophoresis (ODEP) platform provided by the present invention (shown in FIG. 2) uses a digitally projected light image to generate a virtual electrode, which can operate the particles more simply and instantaneously than the metal electrode used in the conventional DEP platform (in the present invention) , microparticles refer to cells, or biological cells).

When cells move on ODEP chips, in addition to DEP forces and resistance, they are subject to several other forces such as buoyancy, gravity, thermal effects, electroosmosis, Brownian motion, and interactions between particles. However, there are many other similar forces that are ignored under the current technical conditions. A normal lymphoma cell is 12 microns in diameter and red blood cells are 6.2-8.2 microns in diameter. In the case of 25Vp-p 60kHz AC, only the gravity and buoyancy and DEP force are among the several forces mentioned above (10). ^-12 N) are of the same order of magnitude, while other orders of magnitude are relatively small, less than 10 ^-15 N. Moreover, the density of the cells is almost equal to the density of the fluid medium, and the cells are suspended in the solution. Therefore, the resultant force of buoyancy and gravity can be ignored. The initial state formula for the movement of cells (mass m, radius R) under DEP forces can be written as:

Where u is the velocity of the cell, F _DEP is the DEP force to which the cell is subjected, F _Viscosity = 6πRηu, and η is the viscosity of the medium. The function of Equation 6 as the speed can be derived:

Where τ _a = m / (6πRη). Since t _a ≈ 10 ^-6 s, and in general the time t is too small to be measured, the accelerated state is usually ignored. Therefore, the velocity formula of the cell under the action of DEP can be simplified as:

Since the DEP force is related to the speed, assuming F _DEP = K × u, where K is the proportional coefficient, then Equation 5 can be expressed as:

According to Equation 8, the velocity u of the velocity particle as a function of time t can be solved:

Where U ₀ is the initial velocity of the particles under the average DEP force, τ = m / (6πRη - K), that is, the time constant. U ₀ and τ are two time-independent constants whose size depends on the physical properties of the electric field and the particles. In this formula, the velocity u(t) varies only with the change of t. In other words, the motion of a particular particle (cell) under a particular DEP field can be represented by a velocity function that decays exponentially over time. Like the other physical properties of the particle, the parameter τ is a time constant that can be measured, indicating the time it takes for the particle to pass a fixed length under a pulsed excitation. Integrating Equation 9 yields the displacement s(t) of the cell (Equation 10), which is derived to obtain the acceleration a(t).

The complete representation of the DEP force that causes particle motion is as follows:

In Equation 8, the DEP force is independent of the position of the particle. From this formula, the distribution of the DEP force on the particle motion trajectory can be derived. The above formula describing the motion of the particles is a function related only to the constants U ₀ and τ.

U ₀ and τ calculated from erythrocyte and lymphoid tumor cell experiments. The results were classified using the Support Vector Machine (SVM) algorithm with an accuracy of 97.5%.

Under the action of DEP, the state of cell movement changes. The image of the cell movement is recorded using a high-speed CCD of an optical microscope to obtain an image sequence I={I _i , i=1, . . . , n}, where n is a time point, and the time of cell movement is determined according to the generation time of the image sequence and the position of the cell. And displacement.

The parameters U ₀ and τ are obtained by fitting the measured sets of displacements and times according to Equation 10. Where U ₀ is the initial velocity of the particles under the average DEP force and τ is the time constant.

To obtain a plurality of sets U ₀ and [tau] of known cell types, the use of these data (plurality of sets of U ₀ and [tau] of known type cells) SVM training.

For an unknown type of cell to obtain a corresponding U ₀ and τ, using the trained SVM classify, you can get cell types.

In summary, the cell sorting method of the present invention has low cost and simple operation, and the entire classification process is basically automated, and the cultured cells can be automatically classified by simply placing the cultured cells into the container, and the classification process is automated. Therefore, the classification of a large number of cells can be completed in a short time, and the efficiency is high. In addition, the method of the present invention has high accuracy and can achieve an accuracy of 97.5% under the existing conditions.

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 cell classification 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: the lower layer is ITO glass coated with a hydrogenated amorphous silicon coating, the upper layer is ITO glass without coating, and the upper and lower layers are ITO. A microfluidic channel is encapsulated between the glass for injecting the 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. Change the frequency and size of the AC signal to control the direction of cell movement while collecting images of the cells;

D. According to the acquired image, the displacement and time of the cells to be classified are fitted, and then the trained support vector machine is used to classify according to the fitting result, and the cell type is obtained.
The cell classification 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. A conductive adhesive is applied to the area of the ITO glass substrate that is not covered with the hydrogenated amorphous silicon coating.
The cell classification 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 cell classification 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 cell classification method based on light-induced 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.
Cell sorting method based on light-induced dielectrophoresis according to claim 5 The method is characterized in that 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.
The cell classification method based on light-induced dielectrophoresis according to claim 3, wherein the displacement formula of the cells under the average dielectrophoretic force is as follows:

U 0 is the initial velocity of the cell under the average dielectrophoretic force, and τ is the time constant.