WO2017028342A1 - 一种基于光诱导介电泳技术的细胞分类方法 - Google Patents

一种基于光诱导介电泳技术的细胞分类方法 Download PDF

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WO2017028342A1
WO2017028342A1 PCT/CN2015/088946 CN2015088946W WO2017028342A1 WO 2017028342 A1 WO2017028342 A1 WO 2017028342A1 CN 2015088946 W CN2015088946 W CN 2015088946W WO 2017028342 A1 WO2017028342 A1 WO 2017028342A1
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cell
light
induced dielectrophoresis
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cells
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李志�
张光烈
李文荣
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Shenzhen University
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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    • G01N27/447—Systems using electrophoresis

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  • the invention relates to the field of cell classification, in particular to a cell classification method based on light-induced dielectrophoresis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • data analysis is difficult to deal with, especially when dealing with low-abundance objects.
  • 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.
  • a cell classification method based on light-induced dielectrophoresis technology comprising the steps of:
  • 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;
  • 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:
  • A2 depositing a hydrogenated amorphous silicon coating on the ITO glass substrate
  • a conductive adhesive is applied to the area of the ITO glass substrate that is not covered with the hydrogenated amorphous silicon coating.
  • F DEP is the average dielectrophoretic force acting on the cell
  • R is the radius of the cell
  • ⁇ 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
  • 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:
  • ⁇ p * and ⁇ m * are the complex dielectric constants of the cells and solutions, respectively.
  • ⁇ is the dielectric constant of the solution
  • ⁇ is the conductivity
  • ⁇ 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:
  • E is the electric field strength
  • ⁇ is the viscosity of the solution
  • IM[f CM ] is the imaginary part of the Clausius-Mossotti factor
  • K is the coefficient
  • U 0 is the initial velocity of the cell under the average dielectrophoretic force
  • ⁇ is the time constant
  • 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.
  • the method of the present invention has high accuracy and can achieve an accuracy of 97.5% under the existing conditions.
  • FIG. 1 is a flow chart of a preferred embodiment of a cell sorting method based on photoinduced dielectrophoresis.
  • FIG. 2 is a schematic view showing the structure of a light-induced dielectrophoresis platform in the present invention.
  • 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.
  • 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:
  • a light-induced dielectrophoresis chip 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;
  • the step of fabricating the light-induced dielectrophoresis chip specifically includes:
  • a layer of hydrogenated amorphous silicon was deposited on the surface of the ITO glass substrate to a thickness of 1 micron.
  • 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 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.
  • 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.
  • a light-induced dielectrophoresis platform is first constructed.
  • 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.
  • optical microscope parameters are as follows:
  • Electric focus can move up and down (upper 13mm / 2mm);
  • Concentrator waterproof, working distance: 7.2mm;
  • Objective lens 20x, highly achromatic lens, nanocrystalline coating
  • Fluorescence filter set FITC/GFP.
  • 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.
  • 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.
  • 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.
  • F DEP is the average dielectrophoretic force acting on the cell
  • R is the radius of the cell
  • ⁇ 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
  • Re[f CM ] is taken when calculating the average dielectrophoretic force, which is defined as follows:
  • Equation 2 ⁇ p * and ⁇ m * are the complex permittivity of the cell and the solution, respectively, and the complex permittivity (including ⁇ p * and ⁇ m *) in Equation 2 can be expressed as:
  • ⁇ is the dielectric constant of the solution
  • ⁇ is the conductivity
  • ⁇ is the frequency of the applied electric field (alternating current signal).
  • 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.
  • E is the electric field strength
  • IM[f CM ] is the imaginary part of the Clausius-Mossotti factor
  • K is the coefficient
  • ⁇ 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.
  • ODEP light-induced dielectrophoretic force
  • 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.
  • 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.
  • ODEP light-induced dielectrophoretic force
  • 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).
  • Equation 6 Equation 6 as the speed can be derived:
  • Equation 5 Equation 5
  • Equation 8 the velocity u of the velocity particle as a function of time t can be solved:
  • U 0 is the initial velocity of the particles under the average DEP force
  • ⁇ m / (6 ⁇ R ⁇ - K)
  • U 0 and ⁇ are two time-independent constants whose size depends on the physical properties of the electric field and the particles.
  • the velocity u(t) varies only with the change of t.
  • the motion of a particular particle (cell) under a particular DEP field can be represented by a velocity function that decays exponentially over time.
  • the parameter ⁇ 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).
  • 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 ⁇ .
  • the state of cell movement changes.
  • the parameters U 0 and ⁇ 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 ⁇ is the time constant.
  • 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.
  • the method of the present invention has high accuracy and can achieve an accuracy of 97.5% under the existing conditions.

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Abstract

一种基于光诱导介电泳技术的细胞分类方法，包括制作由三层结构组成的光诱导介电泳芯片；向所述光诱导介电泳芯片的上下两层ITO玻璃的电极输入可变频率的交流信号，并利用入射光照射所述光诱导介电泳芯片；改变交流信号的频率及大小，以控制细胞运动方向，同时采集细胞的图像；根据采集的图像对待分类细胞的位移和时间进行拟合，根据拟合结果使用已训练的支持向量机来进行分类，得到细胞类型。该分类方法成本低，操作简单，准确率高，能够达到97.5％的准确率。

Description

一种基于光诱导介电泳技术的细胞分类方法 技术领域
本发明涉及细胞分类领域，尤其涉及一种基于光诱导介电泳技术的细胞分类方法。
背景技术
现有技术中，细胞分类主要是以下几种方法：
1、离心法：离心法分类是利用不同尺寸和密度的细胞在离心场中沉降行为的不同，从组织匀浆或血液中将不同细胞分类的技术。用离心技术分离细胞主要依赖的方法是差分离心、速率－区带密度梯度离心、等密度离心和利用特殊转头的细胞浮选离心。
2、膜滤法：用半透膜作为选择障碍层，利用膜的选择性(孔径大小)，以膜的两侧存在的能量差作为推动力，允许某些特定类型的细胞透过而保留其他类型细胞，从而达到分离目的细胞分类技术。
3、荧光激活细胞分类术：即以荧光素标记抗体结合相应细胞，用激光束激发单行流动的细胞，根据细胞所携带的荧光对细胞进行自动分析或分选。其原理是：被荧光染色的细胞在激光束的照射下，产生散射光和激发荧光。光散射信号在前向小角度进行检测，这种信号基本上反映了细胞体积的大小；荧光信号的接受方向与激光束垂直，经过一系列双色性反射镜和带通滤光片的分离，形成多个不同波长的 荧光信号。
但上述方法均存在不足之处：
离心法：成本太高，耗时太久，需要制备介质溶液，操作严格，不易控制。
膜滤法：薄膜的制备过于复杂，不同薄膜对于实验环境的要求不同，薄膜的使用寿命有限。
荧光激活细胞分类术：成本太高，另外存在细胞内低丰度分子难以检测，缺少“通用”细胞通透物质，细胞自发荧光的干扰效应，荧光分子间发射光谱的重叠，以及缺少可识别目标分子的试剂。特别是对于细胞分拣来说，由于液滴的形成收集、分拣细胞重分析或培养前的稀释，以及为获得足够数量的可用细胞需要的时间延误的原因，还存在细胞存活问题。最后，特别是在处理低丰度对象时，数据分析难以处理。
因此，现有技术还有待于改进和发展。
发明内容
鉴于上述现有技术的不足，本发明的目的在于提供一种基于光诱导介电泳技术的细胞分类方法，旨在解决现有细胞分类方法成本高、操作复杂、效率低、准确率低等问题。
本发明的技术方案如下：
一种基于光诱导介电泳技术的细胞分类方法，其中，包括步骤：
A、制作光诱导介电泳芯片，所述光诱导介电泳芯片有三层结构 组成：下层为涂有氢化非晶硅涂层的ITO玻璃，上层是不含涂层的ITO玻璃，在上下两层ITO玻璃之间封装有一个微流体通道，用于注射所需操作的溶液；
B、向上下两层ITO玻璃的电极输入可变频率的交流信号，同时利用入射光照射所述光诱导介电泳芯片，从而在被照射的区域产生非均匀电场；
C、改变交流信号的频率及大小，以控制细胞运动方向，同时采集细胞的图像；
D、根据采集的图像对待分类细胞的位移和时间进行拟合，然后根据拟合结果使用已训练的支持向量机来进行分类，得到细胞类型。
所述的基于光诱导介电泳技术的细胞分类方法，其中，所述步骤A中，制作光诱导介电泳芯片的步骤具体包括：
A1、清理ITO玻璃基质；
A2、在ITO玻璃基质上沉积氢化非晶硅涂层；
A3、在氢化非晶硅涂层上涂光刻胶；
A4、在光刻胶上进行板印；
A5、接触腐蚀至ITO玻璃基质；
A6、去除光刻胶；
A7、在ITO玻璃基质上未覆盖氢化非晶硅涂层的区域涂导电粘合剂。
所述的基于光诱导介电泳技术的细胞分类方法，其中，所述细胞在非均匀电场中的所受到的平均介电泳力用如下公式描述：
Figure PCTCN2015088946-appb-000001
其中FDEP是作用到细胞上的平均介电泳力，R是细胞的半径，εm是细胞所在溶液的介电常数，Erms为所施加交流信号的均方根值，fCM为Clausius-Mossotti因子，在计算平均介电泳力时取该因子的实部Re[fCM]。
所述的基于光诱导介电泳技术的细胞分类方法，其中，fCM因子定义如下:
Figure PCTCN2015088946-appb-000002
εp*和εm*分别是细胞和溶液的复介电常数。
所述的基于光诱导介电泳技术的细胞分类方法，其中，所述复介电常数表示为:
Figure PCTCN2015088946-appb-000003
其中，ε是溶液的介电常数，σ是导电率，ω是所施加交流信号的频率。
所述的基于光诱导介电泳技术的细胞分类方法，其中，细胞旋转速度为：
Figure PCTCN2015088946-appb-000004
其中E是电场强度，η是溶液的黏稠度，IM[fCM]是Clausius-Mossotti因子的虚部，K为系数。
所述的基于光诱导介电泳技术的细胞分类方法，其中，细胞在平均介电泳力下的位移公式如下：
Figure PCTCN2015088946-appb-000005
U0是细胞在平均介电泳力下的初速度，τ是时间常数。
有益效果：本发明的细胞分类方法，其成本低，操作简单，整个分类过程基本上是自动化的，只需把培养好的细胞放入容器中，即可自动分类，由于分类过程的自动化，所以可在很短的时间内完成大量细胞的分类，效率较高。另外本发明的方法准确率高，在现有条件下能够达到97.5％的准确率。
附图说明
图1为本发明一种基于光诱导介电泳技术的细胞分类方法较佳实施例的流程图。
图2为本发明中的光诱导介电泳平台的结构示意图。
具体实施方式
本发明提供一种基于光诱导介电泳技术的细胞分类方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
请参阅图1，图1为本发明一种基于光诱导介电泳技术的细胞分类方法较佳实施例的流程图，如图所示，其包括步骤：
S100、制作光诱导介电泳芯片(ODEP芯片)，所述光诱导介电泳芯片有三层结构组成：下层为涂有氢化非晶硅涂层的ITO玻璃，上 层是不含涂层的ITO玻璃(即不含氢化非晶硅涂层)，在上下两层ITO玻璃之间封装有一个微流体通道，用于注射所需操作的溶液；
S200、向上下两层ITO玻璃的电极输入可变频率的交流信号，同时利用入射光照射所述光诱导介电泳芯片，从而在被照射的区域产生非均匀电场；可先向微流体通道注入细胞和介质(介质即所需操作的溶液，也即细胞所在溶液)。然后输入交流信号。
S300、改变交流信号的频率及大小，以控制细胞运动方向，同时采集细胞的图像；
S400、根据采集的图像对待分类细胞的位移和时间进行拟合，然后根据拟合结果使用已训练的支持向量机来进行分类，得到细胞类型。
进一步，所述的步骤S100中，制作光诱导介电泳芯片的步骤具体包括：
S101、清理ITO玻璃基质；
清理ITO玻璃基质的表面，保证接触面的洁净度。
S102、在ITO玻璃基质上沉积氢化非晶硅涂层(a-Si:H)；
在ITO玻璃基质表面沉积一层氢化非晶硅，厚度为1微米。
S103、在氢化非晶硅涂层上涂光刻胶；
S104、在光刻胶上进行板印；
板印是按照指定图形制作遮盖物，将遮盖物放在光刻胶表面，用紫外线照射遮盖物，没有被遮盖的光刻胶在紫外线作用下溶解，最终得到与遮盖物形状相同的光刻胶层。
S105、接触腐蚀至ITO玻璃基质；具体是用草酸腐蚀制作的芯 片表层，以去除没有覆盖光刻胶的氢化非晶硅涂层。
S106、去除光刻胶；即将光刻胶从氢化非晶硅涂层表面去除。
S107、在ITO玻璃基质上未覆盖氢化非晶硅涂层的区域涂导电粘合剂。即在ITO玻璃的表面没有覆盖氢化非晶硅涂层的位置添加一个导电触点。
而上层的ITO玻璃清理干净之后，涂导电粘合剂即可。
在上下两层ITO玻璃之间封装有一个微流体通道(100微米高)，，具体是通过PDMS或是双面胶封装出一个微流体通道。
在步骤S200中，如图2所示，首先搭建光诱导介电泳平台。除了步骤S100制作的ODEP芯片20，平台还需要一台光学显微镜10、一台光学投影仪(高分辨率)、一个可编程信号发生电路和主机系统。所述主机系统包括：图像采集模块、显微视觉算法处理模块、生物芯片驱动控制器、虚拟电极生成模块以及显示输出模块。所述图像采集模块用来采集光学显微镜20的图像，并交由显微视觉算法处理模块来进行处理并通过显示输出模块来显示，所述显微视觉算法处理模块还向生物芯片驱动控制器及虚拟电极生成模块发出信号用来控制二者工作。所述生物芯片驱动控制器连接所述可编程信号发生电路来改变交流信号频率和大小。所述可编程信号发生电路通过电极连接所述ODEP芯片20。所述光学投影仪设置在ODEP芯片20下方，用来对其进行入射光照射。所述虚拟电极生成模块连接所述光学投影仪。
其中光学显微镜参数如下：
尼康CFI60无限远光学系统；
电动对焦，可上下移动(上13mm/下2mm)；
三目镜筒，光分布：目镜/相机100％/0，20％/100％，0/100％；
目镜放大倍率：10x；
聚光器：防水，工作距离：7.2mm；
物镜：20x，高度消色透镜，纳米结晶涂层；
载物台：电动X轴和Y轴，分辨率：0.1微米；
紫外线截止滤光块；
荧光滤波套装：FITC/GFP。
在平台搭建好后，可通过生物芯片驱动控制器向可编程信号发生电路发出信号，然后可编程信号发生电路向上下两层ITO玻璃的电极输入可变频率的交流信号，同时光学投影仪利用入射光照射所述光诱导介电泳芯片，从而在被照射的区域产生非均匀电场。
在所述步骤S300中，通过改变交流信号的频率及大小，来改变细胞所受到的介电泳力的方向与大小，以控制细胞运动方向，同时采集细胞的图像，实现高速操纵微纳米实体。
下面介绍下，如何实现由改变交流信号的频率及大小来控制细胞运动方向。
细胞在非均匀电场中的所受到的平均介电泳力可以用如下公式描述：
Figure PCTCN2015088946-appb-000006
其中FDEP是作用到细胞上的平均介电泳力，R是细胞的半径，εm是细胞所在溶液的介电常数，Erms为所施加电场(交流信号)的均方 根值，fCM为Clausius-Mossotti因子，在计算平均介电泳力时取该因子的实部Re[fCM]，该因子定义如下:
Figure PCTCN2015088946-appb-000007
εp*和εm*分别是细胞和溶液的复介电常数，公式2中的复介电常数(包括εp*和εm*)可表示为:
Figure PCTCN2015088946-appb-000008
其中，ε是溶液的介电常数，σ是导电率，ω是所施加电场(交流信号)的频率。
可以看出fCM是一个和频率相关的可变因子。考虑在施加不同频率的交变电场下，当介电泳力与电场强度变化方向相同时，称为正介电泳现象；当所受到的介电泳力与电场强度变化方向相反，称为负介电泳现象。因而可以通过改变所施加的电场的频率，来改变细胞所受到的介电泳力的方向，达到控制细胞运动方向的目的。
由于生物细胞受到非均匀电场的极化作用而产生偶极矩，根据其介电泳力所产生的转矩与所在介质中受到的摩擦力矩达到平衡，细胞旋转速度为：
Figure PCTCN2015088946-appb-000009
其中E是电场强度，IM[fCM]是Clausius-Mossotti因子的虚部，K为系数，η是介质(即细胞所在溶液)的黏稠度。根据细胞的旋转速度与细胞的介电常数的关系可以对细胞的介电特性进行估算。
细胞受到的介电泳力强度与方向主要取决于介质与细胞的介电 特性，如形状、尺寸与电场频率。本发明利用光诱导介电泳力(ODEP)(当施加某频段，电液动力学的一种主导力)以识别与操纵生物细胞，分离纳米尺度的聚合物颗粒。ODEP芯片由可变频率的交流信号驱动，交流信号通过上下两层ITO玻璃的导电触点输入，此时在溶液层只有一小部分分压，并在溶液层中产生均匀电场。当入射光照射ODEP芯片，a-Si:H的光导率由于电子空穴对数的增多而增加几个数量级。由于入射光区域电阻减小，在溶液层中的分压会大大增大，于是入射光区域的a:Si:H将成为一个有效的虚拟电极产生非均匀电场。这种光诱导的非均匀电场会极化区域内的颗粒产生介电泳力，也就是光诱导介电泳力(ODEP)。通过光学显微镜与主机系统可实现程序化的动态运动，且不需要任何手工界面而实现微纳米实体的自动化捕获、操纵、分离与组装。因此，本发明的ODEP芯片可实现高速操纵微纳米实体。
介电泳力产生于非均匀电场，而传统的方法需要复杂的制造工艺和准备过程，而且通常会限制细胞的运动。本发明提供的光诱导介电泳(ODEP)平台(如图2所示)使用数字化投射光图像生成虚拟电极，比起普通DEP平台使用的金属电极，能够更加简单、即时的操作微粒(本发明中，微粒指细胞，或生物细胞)。
当细胞在ODEP芯片上运动时，除了DEP力和阻力之外，还会受其他的几个力，如浮力，重力、热效应、电渗透、布朗运动和微粒间的相互作用。但是，在现有的技术条件下还有很多其他类似的力被忽略了。一个正常的淋巴瘤细胞的直径是12微米，红细胞的直径是 6.2-8.2微米，在25Vp-p的60kHz交流电的情况下，前面提到的几个力中，只有重力和浮力与DEP力(10-12N)处于同一数量级，而其他几个力的数量级相对太小，小于10-15N。而且细胞的密度和流体介质的密度几乎相等，细胞悬浮于溶液中。因此，浮力和重力的合力是可以被忽略的。细胞(质量为m，半径为R)在DEP力作用下运动的最初状态公式可以写成：
Figure PCTCN2015088946-appb-000010
其中u是细胞的速度，FDEP是细胞所受的DEP力，FViscosity＝6πRηu，η是介质的黏稠度。可推导出公式6即速度的函数：
Figure PCTCN2015088946-appb-000011
其中τa＝m/(6πRη)。由于ta≈10-6s，而且一般情况下时间t太小而很难被测量，加速态通常被忽略。所以，DEP力作用下细胞的速度公式可简化为：
Figure PCTCN2015088946-appb-000012
由于DEP力与速度相关，假设FDEP＝K×u，其中K是比例系数，则公式5可表示为：
Figure PCTCN2015088946-appb-000013
根据公式8可以解出速度微粒的速度u关于时间t的函数：
Figure PCTCN2015088946-appb-000014
其中U0是微粒在平均DEP力下的初速度，τ＝m/(6πRη-K)，即时间常数。U0和τ是两个与时间无关的常量，它们的大小取决于电场和 微粒的物理性质。在这个公式中，速度u(t)只随着t的变化而变化，换句话说，特定微粒(细胞)在特定DEP场下的运动可以用随时间以指数方式衰减的速度函数表示。同微粒的其他物理性质一样，参数τ是一个可被测量的时间常数，表示微粒在一个脉冲激励下通过固定长度所需要的时间。对公式9积分可以得到细胞的位移s(t)(公式10)，对其求导可以得到加速度a(t)。
Figure PCTCN2015088946-appb-000015
因此造成微粒运动的DEP力的完整表示如下：
Figure PCTCN2015088946-appb-000016
公式8中，DEP力与微粒的位置无关，从这个公式中可以得出微粒运动轨迹上DEP力的分布。上述描述微粒运动的公式都是只与常量U0和τ有关的函数了。
根据红细胞和淋巴肿瘤细胞实验中计算出的U0和τ。使用支持向量机(SVM)算法对结果分类，准确率达到了97.5％。
在DEP力作用下，细胞运动状态发生变化。使用光学显微镜的高速CCD记录细胞运动的图像，得到图像序列I＝{Ii,i＝1,…,n}，n为时刻点，根据图像序列的生成时间和细胞的位置确定细胞运动的时间和位移。
根据公式10对测得的多组位移和时间拟合得到参数U0和τ。其中U0是微粒在平均DEP力下的初速度，τ是时间常数。
先获得多组已知类型细胞的U0和τ，使用这些数据(多组已知类型细胞的U0和τ)训练支持向量机。
对于一个未知类型的细胞，得到对应的U0和τ，使用已训练的支持向量机对其进行分类，即可得到细胞的类型。
综上所述，本发明的细胞分类方法，其成本低，操作简单，整个分类过程基本上是自动化的，只需把培养好的细胞放入容器中，即可自动分类，由于分类过程的自动化，所以可在很短的时间内完成大量细胞的分类，效率较高。另外本发明的方法准确率高，在现有条件下能够达到97.5％的准确率。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (7)

  1. 一种基于光诱导介电泳技术的细胞分类方法，其特征在于，包括步骤：
    A、制作光诱导介电泳芯片，所述光诱导介电泳芯片有三层结构组成：下层为涂有氢化非晶硅涂层的ITO玻璃，上层是不含涂层的ITO玻璃，在上下两层ITO玻璃之间封装有一个微流体通道，用于注射所需操作的溶液；
    B、向上下两层ITO玻璃的电极输入可变频率的交流信号，同时利用入射光照射所述光诱导介电泳芯片，从而在被照射的区域产生非均匀电场；
    C、改变交流信号的频率及大小，以控制细胞运动方向，同时采集细胞的图像；
    D、根据采集的图像对待分类细胞的位移和时间进行拟合，然后根据拟合结果使用已训练的支持向量机来进行分类，得到细胞类型。
  2. 根据权利要求1所述的基于光诱导介电泳技术的细胞分类方法，其特征在于，所述步骤A中，制作光诱导介电泳芯片的步骤具体包括：
    A1、清理ITO玻璃基质；
    A2、在ITO玻璃基质上沉积氢化非晶硅涂层；
    A3、在氢化非晶硅涂层上涂光刻胶；
    A4、在光刻胶上进行板印；
    A5、接触腐蚀至ITO玻璃基质；
    A6、去除光刻胶；
    A7、在ITO玻璃基质上未覆盖氢化非晶硅涂层的区域涂导电粘合剂。
  3. 根据权利要求1所述的基于光诱导介电泳技术的细胞分类方法，其特征在于，所述细胞在非均匀电场中的所受到的平均介电泳力用如下公式描述：
    Figure PCTCN2015088946-appb-100001
    其中FDEP是作用到细胞上的平均介电泳力，R是细胞的半径，εm是细胞所在溶液的介电常数，Erms为所施加交流信号的均方根值，fCM为Clausius-Mossotti因子，在计算平均介电泳力时取该因子的实部Re[fCM]。
  4. 根据权利要求3所述的基于光诱导介电泳技术的细胞分类方法，其特征在于，fCM因子定义如下:
    Figure PCTCN2015088946-appb-100002
    εp*和εm*分别是细胞和溶液的复介电常数。
  5. 根据权利要求4所述的基于光诱导介电泳技术的细胞分类方法，其特征在于，所述复介电常数表示为:
    Figure PCTCN2015088946-appb-100003
    其中，ε是溶液的介电常数，σ是导电率，ω是所施加交流信号的频率。
  6. 根据权利要求5所述的基于光诱导介电泳技术的细胞分类方 法，其特征在于，细胞旋转速度为：
    Figure PCTCN2015088946-appb-100004
    其中E是电场强度，η是溶液的黏稠度，IM[fCM]是Clausius-Mossotti因子的虚部，K为系数。
  7. 根据权利要求3所述的基于光诱导介电泳技术的细胞分类方法，其特征在于，细胞在平均介电泳力下的位移公式如下：
    Figure PCTCN2015088946-appb-100005
    U0是细胞在平均介电泳力下的初速度，τ是时间常数。
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