CN115044468A

CN115044468A - Flow type electric rotating micro device for measuring single cell electric parameter

Info

Publication number: CN115044468A
Application number: CN202210870265.1A
Authority: CN
Inventors: 黄亮; 夏豪杰; 叶海生
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2022-07-18
Filing date: 2022-07-18
Publication date: 2022-09-13

Abstract

The invention relates to the technical field of single cell rotation, in particular to a flow type electric rotation micro device for measuring single cell electric parameters, which comprises: the cell suspension device comprises a substrate, a flow channel body and at least two electrode units, wherein the electrode units are arranged in a line along the flowing direction of the cell suspension; each electrode unit comprises four electrode groups which are distributed in a rectangular mode along the flowing direction of the cell suspension; the four electrode groups of a single electrode unit are respectively applied with sine signals with the same amplitude and the same frequency, the four sine signals have fixed phase difference of 90 degrees along a single winding direction, and a rotating electric field is generated in an area formed by the four electrode groups of the electrode unit; sinusoidal signals applied by two adjacent electrode units along the flowing direction of the cell suspension, wherein the frequencies have difference; the invention realizes multi-frequency point rotation by utilizing the design of a plurality of electrode units, changes the traditional fixed position rotation mode, realizes flow type electric rotation measurement, greatly improves the flux of single cell measurement and reduces the operation complexity.

Description

Flow type electric rotating micro device for measuring single cell electric parameter

Technical Field

The invention relates to the technical field of single cell rotation, in particular to a flow type electric rotation micro device for measuring single cell electric parameters.

Background

In the biomedical field, measurements from a single cell perspective are required, such as in gene analysis, drug development, tissue formation, cancer mechanism, disease treatment, and the like. Due to the high heterogeneity of single cells, characterization of single cells can be achieved by measuring physical and biochemical markers of single cells. Among them, the label-free identification of single cells is of increasing interest to researchers because of its advantages of no damage, low cost, high accuracy, etc. Where the electrical properties of a cell are closely related to its internal structure and chemical properties, this can serve as a unique marker for the cell type. Therefore, measuring the dielectric properties of cells has a great role in the mechanism research of cells, the identification of specific cells and classification, and the like. The measurement of the electrical properties of the single cells enables the current state of the body to be known. This may play a role in the early prevention, diagnosis and monitoring of the course of treatment of the disease.

There are several methods for measuring the intrinsic electrical properties of cells, such as micro-electrical impedance spectroscopy, impedance flow cytometry and electrical rotation. But the accuracy of the electrical rotation technique measurement, and the ability to non-invasively characterize the electrical parameters inside the cell, are superior to other methods.

The existing electric rotation method has the defect that the cell rotation spectrum acquisition flux is low. The inventor has found that the conventional method only adopts a set of rotating electrodes, so that the cells need to be captured in the central region of an electrode chamber for measuring the multi-frequency rotation spectrum, the test is time-consuming, and the throughput of cell measurement is greatly limited due to the dependence on a complex single-cell position control technology.

Disclosure of Invention

In view of the above, there is a need to provide a flow-type electrical rotation micro-device for single-cell electrical parameter measurement, which addresses the above-mentioned problem of low throughput of cell rotation spectrum acquisition.

The invention is realized by adopting the following technical scheme:

the invention provides a flow type electric rotating micro device for measuring single cell electric parameters, which comprises:

a substrate;

a flow channel body having a micro flow channel for flowing a cell suspension at a low speed, the flow channel body being fixed on a substrate; and

the electrode unit is fixed on the substrate, one end of the electrode unit is embedded in the fluid channel body and extends in the micro-channel, and the other end of the electrode unit is an access end for applying an electric signal;

the number of the electrode units is at least two, and the electrode units are arranged in a line along the flowing direction of the cell suspension; each electrode unit comprises four electrode groups which are distributed in a rectangular mode along the flowing direction of the cell suspension;

sinusoidal signals with the same amplitude and the same frequency are respectively applied to the four electrode groups of the single electrode unit, the four sinusoidal signals have fixed phase difference of 90 degrees along a single winding direction, and a rotating electric field is generated in an area formed by the four electrode groups of the electrode unit, so that cells in the rotating electric field rotate;

the frequencies of the sinusoidal signals applied by two adjacent electrode units along the direction of flow of the cell suspension have a difference.

As a still further scheme of the invention: each electrode group comprises a three-dimensional electrode and a plane electrode corresponding to the three-dimensional electrode; all the three-dimensional electrodes are positioned in the micro-channel, one end of each planar electrode is electrically connected with the corresponding three-dimensional electrode, and the other end of each planar electrode extends out of the channel body along the substrate to form a corresponding access end.

As a still further scheme of the invention: the cross section of the three-dimensional electrode is semicircular, and the arc surface side of the three-dimensional electrode is embedded into the flow channel body and extends into the micro-flow channel.

As a still further scheme of the invention: the flow type electric rotating micro device also comprises an electroosmosis movement component which is used for generating electroosmosis flow movement with controllable flow speed by providing direct current voltage with adjustable magnitude so as to drive the cell suspension.

As a still further scheme of the invention: the electroosmosis movement component comprises a positive electrode end and a negative electrode end which are respectively inserted at two ends of a micro flow channel, and the two electrode ends are applied with direct current voltage with adjustable magnitude.

As a still further scheme of the invention: the frequency of the sinusoidal signals applied by the plurality of electrode units along the direction of flow of the cell suspension increases in sequence.

As a still further scheme of the invention: a cell suspension inlet is formed in the top of one end of the flow channel body, and a cell suspension outlet is formed in the top of the other end of the flow channel body;

or/and the flow passage body and the substrate are made of transparent materials;

or/and the runner body and the substrate are bonded in an irreversible manner.

As a still further scheme of the invention: the number of the electrode units is three, and the electrode units are used for forming three rotating electric fields.

The invention also provides a single-cell electrical parameter measurement experimental device, which comprises:

the flow type electric rotating micro device for measuring the electric parameters of the single cell;

a rotation signal control module for applying a sinusoidal signal to the electrode unit;

the electroosmotic flow driving control module is used for providing direct-current voltage with adjustable size;

an image capturing unit for capturing an image of the cell movement state;

and the signal processing unit is used for extracting and processing the data of the image and calculating the single cell electrical parameters.

As a still further scheme of the invention: the signal processing unit extracts the image of the image capturing unit, extracts the cell rotating speed, and then calculates the single cell electrical parameter through the modeling calculation of the neural network model based on the electrical rotation function.

Compared with the prior art, the invention has the following beneficial effects:

the multi-frequency-point rotation is realized by utilizing the design of a plurality of electrode units, the traditional fixed position rotation mode is changed, the flow type electric rotation measurement is realized, the single cell measurement flux is greatly improved, and the operation complexity is reduced.

And 2, realizing a low-speed controllable solution driving mode, providing a measurement basis for the flow type electric rotation measurement method, simultaneously considering flow type movement and high flux test, and ensuring the accuracy of cell rotation speed measurement.

3, the rotating electric field is provided by applying a sinusoidal signal by adopting a three-dimensional electrode with a semicircular cross section, so that the electric field distribution is more uniform and is more beneficial to acting on cells; and measurement errors caused by electric field attenuation are avoided by using a plurality of groups of three-dimensional electrodes, and the measurement precision is improved.

4, the invention utilizes the neural network model based on the electric rotation function to model, and can accurately and quickly extract the single-cell electric parameters according to the multi-frequency point rotation spectrum.

Drawings

FIG. 1 is a schematic diagram of a single-cell electrical parameter measurement experimental apparatus according to a preferred embodiment of the present invention;

FIG. 2 is a schematic diagram of the operation of the flow-type electric rotary micro device of the experimental apparatus of FIG. 1;

FIG. 3 is a block diagram of the flow electric rotary micro device of FIG. 2;

FIG. 4 is a top view of the flow-through electrical rotary micro device of FIG. 3;

FIG. 5 is a front view of the flow-through electrical rotary micro device of FIG. 3;

FIG. 6 is a side view of the flow-through electro-spinning micro-device of FIG. 3;

FIG. 7 is a graph showing the simulation of the electric field at different times during a signal cycle for a single electrode unit of the flow-type electrical rotary micro device of FIG. 3;

FIG. 8 is a graph of simulation of the electric field gradient of a single electrode unit of the flow-through electrical rotary micro device of FIG. 3;

FIG. 9 is a simulation of the flow-through electrical rotary micro device of FIG. 3 between two adjacent electrode units;

FIG. 10 is a simulation graph of the vertical electric field strength of the three-dimensional electrodes of the flow-through electric rotating micro-device of FIG. 3;

FIG. 11 shows the simulation result of the flow-type electrical rotating micro device of FIG. 1 with three electrode units combined with electroosmotic movement;

FIG. 12 is a flow velocity profile of the centerline of the flow channel of FIG. 11;

FIG. 13 is a schematic diagram of the movement of a single cell in the flow-through electro-spinning micro-device of FIG. 1;

FIG. 14 is a block diagram of the neural network model of FIG. 1;

FIG. 15 is a block diagram of the experimental procedure of FIG. 1.

FIG. 16 is a process flow diagram of the flow-through electro-spinning micro-device of FIG. 2;

FIG. 17 is a simulation of the flow-type electrical rotary micro-device of FIG. 1 with only a single electrode unit in combination with electroosmotic movement;

FIG. 18 is a flow velocity profile of the centerline of the flow channel of FIG. 17;

FIG. 19 is a schematic diagram of the principle of dielectrophoresis;

FIG. 20 is a schematic view of the electrical rotation principle;

FIG. 21 shows K for single shell spherical microparticles _CM An imaginary curve;

FIG. 22 is a schematic diagram of a neural network.

In the drawings, the components represented by the respective reference numerals are listed below:

1-substrate, 2-micro flow channel, 201-cell suspension inlet, 202-cell suspension outlet, 301-plane electrode, 302-three-dimensional electrode, 4-single cell.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It will be understood that when an element is referred to as being "mounted on" another element, it can be directly on the other element or intervening elements may also be present. When a component is referred to as being "disposed on" another component, it can be directly on the other component or intervening components may also be present. When an element is referred to as being "secured to" another element, it can be directly secured to the other element or intervening elements may also be present.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "or/and" includes any and all combinations of one or more of the associated listed items.

Example 1

Referring to fig. 1, fig. 1 is a structural diagram of a single-cell electrical parameter measurement experimental apparatus according to a preferred embodiment of the present invention. The single cell electrical parameter measurement experimental device comprises: the device comprises a flow type electric rotating micro device, a rotating signal control module, an electroosmotic flow driving control module, an image capturing unit and a signal processing unit. The flow type electric rotating micro device is used for measuring the electric parameters of single cells. The rotation signal control module is used for applying a sinusoidal signal to the electrode unit. The electroosmotic flow driving control module is used for providing electroosmotic flow driving signals, and the electroosmotic movement assembly outputs direct-current voltage with adjustable size according to the electroosmotic flow driving signals. The image capturing unit is used for capturing images of the cell movement state. The signal processing unit is used for extracting and processing the data of the image and calculating the single cell electric parameters.

Referring to fig. 2 and fig. 3-6, fig. 2 is a schematic diagram of the operation of the flow-type electrical rotating micro device of the experimental apparatus of fig. 1; fig. 3-6 are block diagrams of the flow-through electrical rotary micro device of fig. 2. The flow type electric rotating micro device is a core part for realizing single cell electric parameter measurement of the whole experimental device and comprises a substrate 1, a flow channel body and an electrode unit.

The flow channel body has a micro flow channel 2 for flowing the cell suspension at a low speed, wherein the low speed refers to a flow speed as low as the order of μm/s (see fig. 12 and 18), and can be realized by electroosmotic flow. The runner body is fixed on the substrate 1, and the runner body and the substrate can be bonded in an irreversible mode, for example, a plasma surface treatment mode is adopted, so that the connection tightness of the runner body and the substrate is ensured, and no leakage is ensured.

The electrode unit is also fixed on the substrate 1 and has one end embedded in the fluid channel and extending in the microchannel 2 and the other end being an access end for applying an electrical signal.

The number of the electrode units is at least two, and the electrode units are arranged in a line along the flowing direction of the cell suspension; each electrode unit comprises four electrode groups which are distributed in a rectangular mode along the flowing direction of the cell suspension; the four electrode groups of the single electrode unit are respectively applied with sinusoidal signals with the same amplitude and the same frequency, the four sinusoidal signals have fixed phase difference of 90 degrees along a single winding direction, and a rotating electric field is generated in the area formed by the four electrode groups of the electrode unit, so that cells in the rotating electric field rotate.

The number of the electrode units is selected according to actual needs, the purpose of the electrode units is to perform multipoint measurement, and for convenience of explaining the principle, referring to fig. 2 and 3, in the embodiment, 3 electrode units are adopted; taking fig. 2 and the direction in the drawing as an example, 3 electrode units are arranged in a line along the micro flow channel and divided into a first electrode unit on the left side, a second electrode unit in the middle, and a second electrode unit on the right side; four electrode groups of the first electrode unit are respectively applied with four sinusoidal signals which have the same amplitude, the same frequency and different phases, specifically, as shown in fig. 2, in order of ω along the clockwise direction ₁ 、ω ₁ +π/2、ω ₁ +π、ω ₁ +3π/2，ω ₁ Is the angular frequency of the sinusoidal signal; similarly, the four sinusoidal signals of the second electrode unit have the phase position of omega sequentially along the clockwise direction ₂ 、ω ₂ +π/2、ω ₂ +π、ω ₂ +3 π/2; the phase of four sinusoidal signals of the third electrode unit is omega sequentially along the clockwise direction ₃ 、ω ₃ +π/2、ω ₃ +π、ω ₃ +3π/2。

The formation of the above-mentioned rotating electric field is based on dielectrophoresis techniques. Referring to fig. 7, in a single electrode unit, electric field simulation diagrams at different times in a signal cycle generate a rotating electric field in the electrode unit, and cells in the rotating electric field can be rotated under the action of dielectrophoresis torque; referring to fig. 8, a graph simulating the electric field gradient in a single electrode unit, the electric field gradient in the central region of the electrode is minimized, and thus the cells are converged toward the central region of the electrode by the negative dielectrophoresis force. As shown in fig. 2, the phase difference is fixed at 90 ° in the clockwise direction, and the generated electric field rotates the cell clockwise; if the phase difference is set to-90 °, the direction of the rotating electric field is changed, and the cell can rotate counterclockwise. In addition, the speed of cell rotation is related to the frequency and amplitude of the sinusoidal signal. Thus, by adjusting the parameters of the sinusoidal signal, the direction and speed of cell rotation can be controlled.

It should be noted that four electrode sets are formed between two adjacent electrode units, and an unnecessary electric field coupling effect may be generated. For example, three rotating electric fields are formed by using 3 electrode units in fig. 2; however, four electrodes which are distributed in a rectangular shape are formed between the first electrode unit and the second electrode unit and between the second electrode unit and the third electrode unit.

Therefore, in order to eliminate the above-mentioned influence, the frequencies of the sinusoidal signals applied by the two adjacent electrode units in the direction of flow of the cell suspension have a difference.

Experiments prove that when the frequency difference of sinusoidal signals of two adjacent electrode units is large, a rotating electric field cannot be generated between the two electrode units; specifically, the frequency of the sinusoidal signals applied by the plurality of electrode units along the direction of the cell suspension flow is sequentially increased, and a multiple increasing arrangement mode can be adopted. As can be seen from the simulation result of fig. 9, a regular rotating electric field is not formed between the two electrode units (the frequency f of the signal applied to the left electrode unit is 10kHz, the frequency f of the signal applied to the right electrode unit is 100kHz, and the amplitudes of the voltages applied to the two electrode units are both 5V). While three rotating electric fields are formed corresponding to the above-mentioned 3 electrode units in the present embodiment, the frequency setting may be ω ₃ ＝10*ω ₂ ＝100*ω ₁ (ii) a The multiple relation is adjustable, so that the requirement of measuring the single cell gyrus in a sufficiently wide frequency band is met based on the fact that three frequency intervals can be pulled apartThe speed of rotation.

The sine signal is provided by a rotation signal control module; the rotary signal control module can be connected with the corresponding electrode unit access end by a signal generator through a signal wire.

As shown in fig. 2, each electrode group includes a three-dimensional electrode 302 and a planar electrode 301 corresponding to the three-dimensional electrode 302; all the three-dimensional electrodes 302 are positioned in the micro flow channel 2, one end of the planar electrode 301 is electrically connected with the corresponding three-dimensional electrode 302, and the other end of the planar electrode 301 extends out of the flow channel body along the substrate 1 to form a corresponding access end.

In this embodiment, the cross section of the three-dimensional electrode 302 is semicircular, and the arc surface side of the three-dimensional electrode 302 is embedded in the flow channel body and extends into the micro flow channel 2, so that the generated rotating electric field is distributed more uniformly and has better effect on cells. The present embodiment provides a parametric design of three-dimensional electrodes 302, the radius of the semicircle is 50 μm, the height of the three-dimensional electrodes 302 is 50 μm, the minimum distance between two three-dimensional electrodes 302 is 50 μm, and the electric field of the three-dimensional electrodes is analyzed by simulation, as can be seen from fig. 10, the attenuation of the electric field on the vertical transversal lines at different positions is small, and the electric field strength in the vertical direction is almost unchanged. Therefore, the influence of the height of the cell can be ignored when the electric rotation is carried out, and the accuracy of the rotation speed measurement is improved.

In order to make the cells move in the micro flow channel 2 at a low speed, the flow-type electric rotating micro device of the present embodiment further includes an electroosmotic motion component for driving the cell suspension by supplying a direct voltage with adjustable magnitude to make the fluid in the micro flow channel 2 generate electroosmotic motion with controllable flow rate.

Referring to fig. 2, the electroosmotic movement assembly includes positive and negative electrode terminals respectively inserted at both ends of a microchannel 2, to which a direct current voltage with adjustable magnitude is applied; wherein, the positive and negative electrode ends can be electrode rods or direct lead ends inserted; the direct current voltage with adjustable size can be directly output by an instrument or output by an electroosmotic flow driving control module, for example, a fixed value resistor, an adjustable resistor and the direct current voltage are connected in series to form a loop, the voltage value of the fixed value resistor is inversely related to the resistance value of the adjustable resistor, and the direct current voltage with adjustable size can be provided by adjusting the adjustable resistor.

Wherein, the top of one end of the flow channel body is provided with a cell suspension inlet 201, and the top of the other end is provided with a cell suspension outlet 202, which is used for injecting cell suspension into the micro-flow channel 2 and also used as the insertion position of the positive electrode end and the negative electrode end; the cell suspension inlet 201 and the cell suspension outlet 202 may be relatively large in size, so as to facilitate the dripping of the cell suspension, and facilitate the insertion of positive and negative electrodes for applying a dc voltage.

The electroosmotic flow mode is adopted to drive the cell suspension to flow, and compared with the traditional mode (the existing mode based on a micro-injection pump) has the advantages; in the existing mode, if a conventional low-cost model is adopted, the slow flowing of cells in a small-size flow channel is difficult to realize, and if a precise model is adopted, the price is high, and the flow rate response is slow. The electroosmotic flow is adopted to drive the cell suspension, the direct current voltage of the positive electrode end and the negative electrode end is in direct proportion to the fluid speed, the flow speed is controlled by adjusting the voltage amplitude, and the cell suspension flow speed control device is low in cost and high in controllability.

The driving mode of the cell suspension can also realize the directional movement of the solution by adopting the principle of a communicating vessel, but the effect is not good than the electroosmotic flow mode.

Advantages over conventional approaches: most of the existing electric rotating devices are single-group electrodes, the frequency of applied signals needs to be changed for many times when a plurality of frequency points are measured, and cells are guaranteed to be maintained at fixed positions, namely the center position of a chamber surrounded by the electrodes, so that the flux of the existing structure is low in the aspect of representing the cells, and sample waste is easily caused. The invention designs a plurality of electrode units (for example, the 3 electrode units adopted above), drives the cells to move by using electroosmotic flow, obtains the rotating speed of the cells under different frequencies by applying sinusoidal signals with increasing frequencies on each electrode unit, obtains a multi-point rotating spectrum, and provides a flow type electric rotating measurement method. And the flow rate generated by the mode is far less than the minimum flow rate generated by a conventional injection pump, so that the cells can be ensured to have enough time to complete the rotary motion of a complete period in each rotary electric field, and the accuracy of rotating speed measurement is favorably ensured. Referring specifically to FIG. 11, which is a simulation result of the three electrode units arranged in combination with electroosmotic movement (the electric field intensity between the electrodes is 1000V/m), FIG. 12 is a flow velocity situation of the center line of the flow channel in FIG. 11, and the mobility of the cell suspension is accelerated when passing through the electrode area. FIG. 13 shows that the single cell 4 moves in the micro flow channel, and the cell rotates while moving through the rotating electric field region.

According to the experimental device for measuring the single-cell electrical parameters, the flow-type electrical rotation micro device, the rotation signal control module and the electroosmotic flow driving control module are used together, so that cells move along the micro channel 2 and rotate in a plurality of rotating electric fields;

then, the moving state of the cells is imaged through an imaging unit, and referring to fig. 1, the imaging unit can adopt a large-view eyepiece to acquire moving state images of the cells flowing through a plurality of rotating electric fields; the runner body and the substrate 1 are made of transparent materials, so that sampling is convenient.

And then the signal processing unit extracts and processes the image data to calculate the single cell electrical parameters. Specifically, the signal processing unit extracts the image of the image capturing unit, extracts the cell rotation speed, and then calculates the single cell electrical parameter through the electrical rotation function-based neural network model modeling calculation. Fig. 14 is a structural diagram of a neural network model, in which an input layer is a plurality of sets of rotation speeds corresponding to cells passing through a plurality of rotating electric fields, and two electrical parameters required for output from an output layer are output after fitting of the neural network model (electrical rotation function) passing through a hidden layer. In order to obtain effective and accurate fitting parameters and reduce overfitting phenomena, the arrangement mode of the flow type electric rotating micro device is adopted, 3 electrode units are arranged, and three rotating electric fields are formed to obtain three groups of rotating speeds (omega) ₁ 、Ω ₂ 、Ω ₃ ) And inputting the values, and finally outputting the cell dielectric constant epsilon and the cell conductivity sigma. The process utilizes a neural network algorithm and accurately and quickly extracts single-cell electrical parameters through multi-frequency point rotation spectrums.

Referring to fig. 15, a specific step chart of the experiment includes:

1. cell feeding: the cell suspension enters through the cell suspension inlet 201, flows through the electrically rotating region by electroosmosis, and exits through the cell suspension outlet 202.

2. Cell driving: when a cell suspension is introduced into the micro flow channel 2, a voltage is applied to the cell suspension inlet 201 and the cell suspension outlet 202, and the solution flows by the electric field and finally flows to the cell suspension outlet 202.

3. Cell rotation: after the cells flow through the electric rotating area, the cells can generate rotating motion by applying electric signals with the same frequency, the same amplitude and different phase differences on the electrode units; the direction of rotation is related to the order of the phase differences of the electrical signals. The speed of rotation is related to the frequency and amplitude of the electrical signal. Therefore, the direction and speed of cell rotation can be controlled by adjusting the parameters of the electrical signal.

4. And (3) cell recovery: after the cells are subjected to the electrical spinning operation, the cells are recovered at the cell suspension outlet 202.

5. Obtaining cell electrical parameters: and sampling the cell motion state, extracting the rotating speed to obtain a rotating spectrum, and calculating by matching with a neural network model to obtain characteristic parameters.

The following is a supplementary explanation of the design principle of the above structure:

(a) dielectrophoretic force

Polarizable particles (cells) are polarized in a non-uniform electric field to form electric dipoles, and a dielectrophoretic force F is generated under the action of the electric field _DEP Or torque, the schematic diagram is shown in fig. 19.

Wherein the content of the first and second substances,

K _CM is a Clausius-Moxoti factor, which represents the frequency response of the particle to an external electric field and can characterize the size, composition, structure, and surface charge and concentration of the particle. K _CM The magnitude is not only related to the dielectric constant but also to the frequency of the applied signal, the sign of which determines the direction of dielectrophoresis experienced by the particle. R is the radius of the particle (cell),

is the gradient of the square of the electric field, ε _m Is the dielectric constant of the solution;

ε* _m and ε _c Complex permittivity of solution and cells, ε is permittivity, j is the imaginary part of the complex number, ω is angular frequency, σ is conductivity, σ is the electrical conductivity, and _m and σ _c The conductivity, dielectrophoretic force F of the solution and the cells, respectively _DEP The size and the direction are same as K _CM The real part of the coefficients is related.

Dielectrophoretic force F _DEP Is the gradient of the radius R of the particle and the square of the electric field

In direct proportion, when K _CM The real part of the coefficient is positive, the dielectrophoresis is positive dielectrophoresis pDPEP, the dielectrophoretic force will pull the cell to the place with high electric field gradient, when K is _CM When the real part of the coefficient is negative, the dielectrophoretic force is negative dielectrophoretic nDEP, which pulls the cell towards a place where the electric field gradient is low.

(b) Dielectrophoretic rotation torque

The non-uniform electric field generates a torque on the particle, causing it to spin. When a single cell is located among the four electrodes, and signals with the same amplitude, the same frequency and different phases are respectively applied to the four electrodes, a rotating electric field is formed at the central position to generate dielectrophoresis torque to promote the effective rotation of the cell, and the principle of electric rotation is shown in fig. 20.

Γ _ROT ＝-4πR ³ ε _m Im[K _CM ] ^E 2

I.e. dielectrophoretic torque Γ _ROT And the square of the electric field E ₂ 、K _CM Has a relationship to the imaginary part of; k _CM The magnitude of the imaginary part is related to the frequency of the electric field, and the direction of cell rotation can be changed by changing the phase difference of the four electrodes, namely the rotation direction of the electric field.

K of single-shell spherical particles _CM The imaginary part curve is shown in FIG. 21;

the rotating process of the cells in the suspension medium can be subjected to the Stokes force dragging force gamma _f ：

Γ _f ＝8πηΩR ³

Where Ω is the angular velocity of rotation, R is the radius of the cell, and η is the viscosity of the solution.

When the rotational torque and the stokes drag are balanced, i.e.:

|Γ _ROT |＝|Γ _f |

the cells will rotate at a constant speed, and the corresponding angular velocity can be expressed as:

from the above formula, it can be known that the rotation angular velocity of the cell in the rotating electric field is related to various dielectric parameters of the cell, and therefore, the value of the dielectric parameter of the cell can be fitted according to the electrical rotation motion law of the cell and the numerical relationship between the rotation angular velocity and the dielectric parameter.

(c) Electroosmotic flow

The control formula of electroosmotic flow consists of a fluid formula and an electric field formula. Wherein the electric field comprises an electric field generated by an applied vertical electric field phi and an electric double layer Zeta potential Zeta.

Where Ψ is a potential due to zeta potential,

is the square of the gradient; ε is a dielectric constant;c ₀ is the molar concentration away from the diffusion layer; f is a Faraday constant; z is the ionic valence of the solution; k is a radical of _b Boltzmann constant; e is the base charge; t is the temperature.

The fluid formula is a navier-stokes equation:

wherein upsilon is a flow velocity,

is the square of the gradient; t is time; ρ is the fluid density; p is the pressure intensity; μ is the hydrodynamic viscosity; f is the volume force of the electric field acting on the unit mass of the fluid after gravity is omitted.

The velocity of electroosmotic flow is related to the applied electric field and Zeta potential and can be expressed by the following formula:

wherein, mu _EO Is the electroosmotic flow velocity in the channel; epsilon _r Is a relative dielectric constant; epsilon ₀ Is a vacuum dielectric constant; zeta is a Zeta potential; e is the electric field strength and μ is the hydrodynamic viscosity.

(d) Neural network

Neural Networks (Neural Networks) are an important machine learning technology, and related algorithms are inspired by neuron structures and functions. The neuron cell is divided into a cell body and a protrusion, and has the functions of communicating, integrating input information and transmitting information. The processes include both dendrites, which receive impulses transmitted from axons of other neuronal cells and transmit them to the cell body, and axons, which receive external stimuli and process the impulses from the cell body. The neural network is based on the above and mainly comprises an input layer, an output layer and a hidden layer, wherein the input layer is responsible for receiving data needing to be processed, the output layer integrates and outputs the processed data, and the hidden layer is positioned between the input layer and the output layer. Each layer of the network is composed of a plurality of neurons, and each neuron has a weight for adjusting received data. All neurons in the previous layer are multiplied by respective weights, summed and biased, and then processed by an activation function to serve as the output of the neuron in the previous layer, and a schematic diagram of the principle is shown in fig. 22.

The mathematical model corresponding to the neuron is:

wherein, y _k Outputting a matrix for the neural network; x is the number of _i Inputting data;

an activation function for the current layer; omega _ki The weight of the ith neuron of the current layer; b is a mixture of _k Is the bias of the current layer;

transpose for the current layer neuron weight matrix; x is an input matrix; b is a bias matrix.

The use of activation functions can add non-linear factors to make the number of layers in the network meaningful. The activation function is therefore generally non-linear, conductive and computationally simple. Commonly used activation functions are Sigmoid function, tanh function and ReLu function. The loss function is used to evaluate how different the predicted and actual values of the model are. Therefore, the choice of the loss function also has a certain influence on the performance of the model. Commonly used loss functions include absolute value loss function, mean square error function, and cross entropy loss function. Where cross entropy can be used to measure the difference between two probability distributions, cross entropy is often used as a loss function in classification problems. After the number of network layers, the number of neurons in each layer, an activation function, a loss function, training times and other hyperparameters are determined, the set network is trained by using the existing data, namely, the values of all weights are adjusted to be optimal, and the parameters in the network are adjusted according to the training result of each time, so that the data fitting and predicting effects of the whole network are optimal. Selecting 70% of data as a training set for debugging a neural network; selecting 20% as a verification set for checking the training effect; the remaining 10% of the data was used as the validation set; for testing the actual learning capabilities of the network. Neural networks have the advantage of rapidly analyzing complex input data with higher accuracy, which makes it possible to play a significant role in the field of characterizing cells and classifying in microfluidic technology.

Example 2

The invention also provides a manufacturing method of the flow-type electric rotating micro-device in the embodiment 1, as shown in fig. 13. The device is processed into an upper part and a lower part, wherein the upper layer flow channel is manufactured to comprise a three-dimensional electrode, firstly, photoresist (such as SU-8 negative photoresist) is patterned to form a flow channel through a soft lithography technology, then, a mixture (mass ratio is 1:3) of conductive nano particles (such as conductive carbon powder, nano silver powder and the like) and PDMS (polydimethylsiloxane) is manually coated on a mold, and a blade is used for slightly scraping redundant conductive mixture. After the curing by baking, pure PDMS was introduced again and cured by baking again. Finally, demoulding and punching are carried out to finish the manufacture of the upper layer. The lower layer is manufactured by manufacturing a patterned transparent ITO (indium tin oxide) electrode (namely a planar electrode), patterning a photoresist (such as BN303 negative photoresist) by using a soft lithography technology, and etching the ITO by using wet etching or dry etching to finish the manufacturing of the ITO electrode. After the upper layer and the lower layer are manufactured, plasma surface treatment can be adopted for irreversible bonding, and finally, the conducting wire and the ITO electrode are electrically connected (practical conducting adhesive tape, conducting silver adhesive and the like).

The three-dimensional electrode can also be made of conductive silver adhesive, liquid metal and other materials by combining a micro-processing technology; the planar electrode can be made of materials such as metal electrodes and the like by combining a micro-processing technology.

In addition, in the design process of the present invention, simulation was also performed on the conventional method in which only a single electrode unit was provided (the electric field strength between the electrodes was 1000V/m), referring to fig. 17 and 18. The voltage is applied to the cell suspension inlet and outlet at two ends, the flow velocity caused by electroosmosis is relatively uniform, and when the cell suspension flows through the electrode unit area, the width is reduced, and the solution flow velocity is accelerated.

Example 3

The invention also provides a mode for establishing the neural network to settle the cell point parameters based on the electric rotation theory. The neural network establishes a set of models for processing complex data according to the characteristics of the neurons, and extracts the characteristics of the existing data by training the existing marked data.

The invention sets three groups of frequency points, namely three pairs of input signals, and the number of the neurons of the input layer is three. For digital data to be processed in the invention, the number of layers of the hidden layer is set to three, so that not only can effective and accurate fitting parameters be realized, but also the overfitting phenomenon can be reduced. The number of neurons in the output layer is set to be two, namely the predicted values of two parameters are input. When the requirements are fitting parameters (permittivity epsilon of the cell, conductivity sigma of the cell), the Mean Square Error (MSE) of the real and predicted values is chosen as the loss function. In the invention, an electric rotation theoretical formula is packaged into an electric rotation function; the function contains two unknown parameters (epsilon, sigma), which are given by the output layer according to the result of each training, and other parameters are known parameters. And predicting the electric rotation function. By selecting the mean square error as a loss function, a fitting value having a small difference from the actual parameter can be trained.

The function of the Mean Square Error (MSE) is as follows:

wherein, Y _i In order to obtain the data obtained for the experiments,

for the data output by the output layer of the neural network, the mean square error is taken as a loss function, and the value of the loss function can be reduced by adjusting the structure or parameters of the network, so that the fitting error is reduced.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A flow-through electrical rotary micro-device for single cell electrical parameter measurement, comprising:

a substrate (1);

a flow channel body having a micro flow channel (2) for flowing a cell suspension at a low speed, the flow channel body being fixed on a substrate (1); and

the electrode unit is fixed on the substrate (1), one end of the electrode unit is embedded in the fluid channel body and extends in the micro-channel (2), and the other end of the electrode unit is an access end for applying an electric signal;

the cell suspension flow meter is characterized in that the number of the electrode units is at least two, and the electrode units are arranged in a line along the flow direction of the cell suspension; each electrode unit comprises four electrode groups which are distributed in a rectangular mode along the flowing direction of the cell suspension;

2. A flow-type electrical rotary micro-device for single-cell electrical parameter measurement according to claim 1, characterized in that each set of electrodes comprises one three-dimensional electrode (302) and one planar electrode (301) corresponding to said three-dimensional electrode (302); all the three-dimensional electrodes (302) are positioned in the micro-channel (2), one end of the planar electrode (301) is electrically connected with the corresponding three-dimensional electrode (302), and the other end of the planar electrode (301) extends out of the channel body along the substrate (1) to form a corresponding access end.

3. The flow-type electric rotating micro-device for single-cell electric parameter measurement as claimed in claim 2, wherein the cross section of the three-dimensional electrode (302) is semicircular, and the arc side of the three-dimensional electrode (302) is embedded in the flow channel body and extends in the micro-flow channel (2).

4. The flow-type electric rotating micro-device for single-cell electric parameter measurement according to claim 1, further comprising an electroosmotic motion component for driving the cell suspension by providing an adjustable-magnitude DC voltage to generate a controllable-flow-rate electroosmotic motion of the fluid in the micro flow channel (2).

5. The single-cell electrical parameter measurement flow-type electrical rotary micro-device according to claim 4, wherein the electroosmotic movement component comprises positive and negative electrode terminals respectively inserted at two ends of the micro flow channel (2), and the two electrode terminals are applied with direct current voltage with adjustable magnitude.

6. The flow-type electric rotating micro-device for single-cell electric parameter measurement as claimed in claim 1, wherein the frequency of the sinusoidal signal applied by the plurality of electrode units along the direction of cell suspension flow increases sequentially.

7. The flow-type electric rotating micro-device for single-cell electric parameter measurement according to claim 1, wherein the flow channel body is provided with a cell suspension inlet (201) at the top of one end and a cell suspension outlet (202) at the top of the other end;

or/and the flow channel body and the substrate (1) are made of transparent materials;

or/and the runner body and the substrate (1) are bonded in an irreversible manner.

8. The flow-type electric rotating micro-device for measuring the electric parameter of the single cell as claimed in any one of claims 1 to 7, wherein the number of the electrode units is three, and three rotating electric fields are formed.

9. The utility model provides a unicellular electrical parameter measurement experimental apparatus which characterized in that: the method comprises the following steps:

a flow-through electrical rotary micro-device for single-cell electrical parameter measurement as claimed in any one of claims 1-8;

an image capturing unit for capturing an image of the cell movement state;

10. The experimental apparatus for measuring single-cell electrical parameters as claimed in claim 9, wherein the signal processing unit extracts the image of the image capturing unit, extracts the cell rotation speed, and then calculates the single-cell electrical parameters by modeling and calculation based on the electrical rotation function neural network.