CN104251810A - System for simultaneous representation of single cell Young's modulus and cell membrane specific capacitance - Google Patents

Abstract

The invention provides a system for simultaneous representation of single cell Young's modulus and cell membrane specific capacitance based on a microfluidic technology. The system makes a cell equivalent to an isotropic super-viscoelastic body, based on ABAQUS mechanical simulation, the relation between the displacement generated by instantaneous entrance of the cell front end into a compression channel, and the cell size, the Young's modulus, the pressure intensity, and geometric parameters of the compression channel, and the Young's modulus of the cell can be calculated. In addition, the invention also puts forward an equivalent electrical model for the cell to pass through the compression channel, the relation between the cell membrane capacitance, cytoplasm resistance, cell and compression channel wall leakage resistance, electrical parameters of the compression channel itself, and the impedance spectrum can be obtained, the impedance change caused by passing of the cell through the compression channel is converted to cell membrane specific capacitance, so that the cell membrane specific capacitance of the single cell can be calculated, thus realizing synchronous measurement of the single cell Young's modulus and cell membrane specific capacitance.

Description

Characterize the system of unicellular Young modulus and cell membrane ratio capacitance simultaneously

Technical field

The present invention relates to biological information detection field, particularly relate to a kind of system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance.

Background technology

Cell is as the base unit of vital movement, and containing various biomolecule, their interphase interaction, common forms a busy and orderly system.The mechanics parameters of cell and Young modulus, determined by cytoskeleton; The electrology characteristic parameter of cell and cell membrane ratio capacitance (electric capacity of cell membrane unit area), be made up of phospholipid bilayer and memebrane protein.Cytoskeleton and cell membrane, as the critical function unit of cell, participate in the physiological function that cell proliferation, division etc. are important, closely related with cell state.Although traditional characterization technique such as atomic force microscope, patch-clamp etc. can the Young modulus of preliminary characterization cell and cell membrane ratio capacitance, but above-mentioned technology for detection flux is low and cannot characterize mechanics and the electrology characteristic of cell, the sign of restrictive cell biophysical properties simultaneously.

Micro-fluidic chip is called as " chip lab ", is the novel solid element made by semiconductor integration technology, can carries out complexity, operate accurately micro fluid.Due to the characteristic dimension of micro-fluidic chip and cell size comparable, be suitable for single celled catching and characteristic present, be tentatively used for gathering the mechanics/electrology characteristic of cell simultaneously.

The Prof.Fujii seminar of Tokyo Univ Japan in 2006 proposes the semi-girder sequence capturing individual cells based on microflow control technique, semi-girder electrode is used to characterize the electrology characteristic of captured cell, simultaneously according to the mechanical characteristic causing the deformation extent of semi-girder to characterize individual cells in cell capture process.This seminar uses this method successfully to distinguish normal and ill red blood cell.

Realizing in process of the present invention, applicant finds that the method that existing cyto-mechanics/electrology characteristic high flux synchronously characterizes exists following defect: namely can only characterizing some biophysical properties parameters depending on cell size as entered the time, semi-girder degree of deformation, impedance spectrum etc. of pressure channel, can not characterize Young modulus and the cell membrane ratio capacitance of cell intrinsic mechanics/electrology characteristic parameter and cell.

Summary of the invention

(1) technical matters that will solve

In view of above-mentioned technical matters, the invention provides a kind of system based on characterizing unicellular Young modulus and cell membrane ratio capacitance while microflow control technique, synchronously characterizing with the high flux realizing individual cells Young modulus and cell membrane ratio capacitance.

(2) technical scheme

The invention provides a kind of system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance.This system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance comprises: micro-fluidic chip, image detection module, impedance information acquisition module and data processing module.

Micro-fluidic chip, comprising: transparent substrates, supporting body, is formed at described transparent substrates front; And pressure channel, be formed at described supporting body inner, its lower surface is the upper surface of described transparent substrates, and for passing through for cell compression to be measured, the cross-sectional area of this pressure channel is less than the cross-sectional area of described cell to be measured.

Image detection module, for taking cell to be measured process by described pressure channel under suction function from the lower surface of described transparent substrates.

Impedance information acquisition module, its two potential electrode stretches into the both sides of described pressure channel, for recording compressed passage both sides, the time dependent waveform of impedance of corresponding low frequency and high frequency two Frequency points.

Data processing module, is connected with impedance information acquisition module impedance information acquisition module with described image detection module, comprises: Young modulus obtains submodule, and the image for being taken by camera obtains the transient Displacements X that cell to be measured enters pressure channel _{instantaneous}with unstability displacement X _transitional, and then in conjunction with the diameter d of cell to be measured _cell? _diameter, the logical size W of compression _constriction? _channel, negative pressure numerical value P _aspirationcalculate the Young modulus of cell to be measured; Cell membrane ratio capacitance obtains submodule, obtains for the image taken by camera the length L being in the cell under extended state in pressure channel _cell? _elongation, extract in the waveform obtained by electric impedance analyzer do not have cell by time pressure channel two ends low-frequency impedance Z _l0(ω) with high-frequency resistance Z _h0(ω), have cell by time pressure channel two ends low-frequency impedance Z _l1(ω), high-frequency resistance Z _h1, and then calculate the film ratio capacitance of cell to be measured (ω).

(3) beneficial effect

As can be seen from technique scheme, the system that the present invention characterizes unicellular Young modulus and cell membrane ratio capacitance simultaneously has following beneficial effect:

(1) cell is equivalent to isotropic super viscoelastic body, based on ABAQUS Mechanics Simulation obtain cell front end instantaneous enter pressure channel displacement and the relation of geometric parameter of the size of cell, Young modulus, pressure and pressure channel, calculate the Young modulus of cell;

(2) equivalent electrical model of cell by pressure channel is proposed, obtain cell membrane capacitance, tenuigenin resistance, cell and the electrical parameter of pressure channel wall leakage resistance and pressure channel self and the relation of impedance spectrum, cause the change of impedance to be converted into cell membrane ratio capacitance by pressure channel in cell, thus calculate single celled cell membrane ratio capacitance;

(3) the present invention proposes to characterize the Young modulus of cell and the method for cell membrane ratio capacitance simultaneously, the high flux of the Young modulus and cell membrane ratio capacitance that realize the intrinsic mechanics of cell and electrology characteristic parameter and cell gathers simultaneously, and the sign for cell biological physical characteristics provides reliable method and access;

(4) micro-fluidic chip used is chosen the lower cost materials such as microslide and dimethyl silicone polymer and is processed, based on fine machining method, have cost low, can mass manufacture, the feature such as disposable;

Comprehensive above-mentioned advantage, the present invention can be the disease that anaemia, tumour etc. exist the corresponding change of cell biological physical characteristics and provides new detection means and the new cell characteristics mark without the need to mark.

Accompanying drawing explanation

Fig. 1 is the structural representation of the system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance according to the embodiment of the present invention.

Fig. 2 is the process flow diagram of micro-flow control chip preparation method in system described in Fig. 1;

Fig. 3 is that certain cell of application example collection of the present invention enters the picture of pressure channel under the effect of the pressure and measures and obtain cell front end and carry out the displacement of pressure channel and the relation of time;

Fig. 4 is that application example of the present invention gathers cell and causes the change of double frequency impedance by pressure channel and be arranged in the image of certain cell of pressure channel;

Fig. 5 the present invention is based on the relation of Young modulus that cell that computer simulation obtains enters the transient Displacements of the simulation result of pressure channel and cell, unstability displacement and cell;

Fig. 6 is the equivalent electrical model schematic diagram of pressure channel in varied situations in the present invention.

Embodiment

For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.It should be noted that, in accompanying drawing or instructions describe, similar or identical part all uses identical figure number.The implementation not illustrating in accompanying drawing or describe is form known to a person of ordinary skill in the art in art.In addition, although herein can providing package containing the demonstration of the parameter of particular value, should be appreciated that, parameter without the need to definitely equaling corresponding value, but can be similar to corresponding value in acceptable error margin or design constraint.The direction term mentioned in embodiment, such as " on ", D score, "front", "rear", "left", "right" etc., be only the direction with reference to accompanying drawing.Therefore, the direction term of use is used to illustrate and is not used for limiting the scope of the invention.

The invention provides one and realize the system that the intrinsic mechanical characteristic of individual cells (i.e. Young modulus) and electrology characteristic (i.e. cell membrane ratio capacitance) high flux synchronously characterize.

In one exemplary embodiment of the present invention, provide a kind of system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance.Fig. 1 is the structural representation of the system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance according to the embodiment of the present invention.Please refer to Fig. 1, the system that the present embodiment characterizes unicellular Young modulus and cell membrane ratio capacitance simultaneously comprises: micro-fluidic chip, image detection module, impedance information acquisition module and data processing module.

Below respectively each ingredient that the present embodiment characterizes the system of unicellular Young modulus and cell membrane ratio capacitance is simultaneously described in detail.

Please refer to Fig. 1, micro-fluidic chip, comprising: transparent glass substrate; Supporting body, its material is dimethyl silicone polymer, and it is formed at described transparent substrates front; And the pressure channel that the confession being formed at supporting body inside cell compression to be detected passes through, the cross-sectional area of this pressure channel is less than the cross-sectional area of cell.Cell to be measured is out of shape and pushes through pressure channel under the effect of negative pressure, effectively stops electric field line, reduces leakage current.

In the present embodiment, in addition to a glass substrate, the transparent plastic materials such as dimethyl silicone polymer can also be adopted to be used as substrate; Except dimethyl silicone polymer, can also adopt organic glass, the transparent plastic material such as SU8 carrys out injection moulding and forms above-mentioned supporting body.

Pressure channel, be formed at the bottom of supporting body, its lower surface is the upper surface device of described glass substrate, and its xsect is square, and this foursquare cross-sectional area is about the 40%-90% of cell cross section to be measured long-pending (being about 110-250 square micron).

Except pressure channel, above-mentioned supporting body is also formed as lower part: two sections of cell introduction passages, are formed at the left and right sides of pressure channel on supporting body respectively, its xsect is similarly square, and size is greater than the cross sectional dimensions of described pressure channel; Sample injection port, extends to left side cell introduction passage, for injecting the sample comprising cell to be measured by supporting body upper surface; Sample suction outlet, right side cell introduction passage is extended to by supporting body upper surface, its upper end is connected to gas pressure regulator by airtight flexible pipe, for providing negative pressure that cell is drawn through pressure channel by gas pressure regulator, thus makes cytomorphosis to be measured by described pressure channel.

In the present embodiment, the diameter d of cell to be measured _{cell-diameter}be about 15.6 μm, accordingly, the xsect of pressure channel is square, its length of side W _constriction? _channelbe 10 μm, the height of the sample cell at pressure channel two ends is 45 μm.Via experiment, determine that the negative pressure drawing cell is 500 handkerchiefs.

Micro-fluidic chip is the double-deck pressure channel based on dimethyl silicone polymer, adopts fine process to make.Fig. 2 is the process flow diagram of micro-flow control chip preparation method in system described in Fig. 1.As shown in Figure 2, the preparation process of this micro-fluidic chip is as follows:

Step S202, microslide (75 millimeters long, 25 mm wides and 1 millimeters thick) cleans in acetone, ethanol and deionized water, dry (150 DEG C, 30 minutes), one deck SU-85 (1500RPM is applied in microslide Rotating with Uniform, 35 seconds), as shown in (A) in Fig. 2;

Step S204, on microslide SU-85 photoresist front baking (65 DEG C, 2 minutes; 95 DEG C, 5 minutes), exposure (60mW/cm ²), as shown in (B) in Fig. 2, do not develop, and rear baking (65 DEG C, 1 minute; 95 DEG C, 1 minute), thus form pressure channel formpiston;

Step S206, on exposed SU-85 photoresist, Rotating with Uniform applies one deck SU-825 photoresist (2000RPM, 35 seconds) again, as shown in (C) in Fig. 2;

Step S208, to above-mentioned SU-825 photoresist carry out front baking (65 DEG C, 3 minutes; 95 DEG C, 7 minutes), after being aimed at the image that SU-85 exposes by the image in photo mask board, (75mW/cm is exposed to SU-525 photoresist ²), as shown in (D) in Fig. 2;

Step S210, to exposure after SU-825 photoresist after dry (65 DEG C, 1 minute; 95 DEG C, 3 minutes), development (120 seconds);

Step S212, carries out perpendicular film (175 DEG C, 2 hours) to described SU-85 and SU-825 photoresist, forms cell introduction passage formpiston, as shown in (E) in Fig. 2;

Step S214, then at performed polymer and the hardening agent (10: 1) of the liquid dimethyl silicone polymer of mould upper, as shown in (F) in Fig. 2;

Step S216, solidification (120 DEG C, 2 hours) the afterwards demoulding obtains microfluidic channel, as shown in (G) in Fig. 2;

Step S218 is finally dimethyl silicone polymer punching, as shown in (H) in Fig. 2;

Step S220, realizes itself and glass substrate sealing-in, completes micro-fluidic chip, as shown in (I) in Fig. 2.

Above-mentioned preparation technology, for the technician being engaged in micro-fluidic chip field, by the description of above-mentioned preparation process, and by reference to the accompanying drawings, very clearly can understand the structure of this micro-fluidic chip, repeat no more herein.

Image detection module is for taking cell to be measured process by described pressure channel under suction function.Please refer to Fig. 1, this image detection module comprises: image-forming component is aimed at the biological inverted microscope of described pressure channel and aimed at the camera of biological inverted microscope from the back side of transparent glass substrate, wherein, be used for small channel image and cell image etc. to be converted to can the image size of camera-shot for described biological inverted microscope; Camera is used for being out of shape under suction function by the image information in described pressure channel process by biological inverted microscope record cell.

In the present embodiment, microscopical enlargement factor is 400 times, and the sweep velocity of camera is 200 frames/second.Be 20 collection points per second for gathering the sample frequency of the electric impedance analyzer of impedance operator, survey frequency is 1kHz and 100kHz.

The present embodiment in use, use negative pressure that cell is drawn through pressure channel continuously, image detection module real time record cell its front end under suction function is extended gradually and to be entered and by the physical process (scheming in as Fig. 3 shown in A ~ figure D) of pressure channel.By (A) in Fig. 3 ~ (D), cell can be obtained and extend through in pressure channel process gradually, front end relative to the time dependent curve of the displacement of entrance, as shown in (E) in Fig. 3.Just can show that cell to be measured enters instantaneous displacement, the unstability displacement of pressure channel by this curve.

Wherein, cell to be measured, under suction function, is inhaled in pressure channel instantaneously, produces certain displacement immediately, the immediate movement of this displacement and cell to be measured.Because cell belongs to viscoelastic body material, in the moment being subject to External Force Acting, elasticity plays a major role, and produces obvious deformation, i.e. immediate movement, and namely cell is in the displacement in zero positive moment.Due to the cause of pressure channel shape, cell is entering in pressure channel process, when entering into certain displacement, the power moment that suffered resistance and negative pressure apply is uneven, produce very large acceleration change, be referred to as unstability displacement in the displacement of the relative pressure channel entrance in this Place cell front end.By above-mentioned instantaneous displacement and unstability displacement, just can calculate the Young module of cell to be measured in conjunction with information such as cell dia, the pressure channel length of sides, will be described in more detail below.

Impedance information acquisition module, its two potential electrode stretches into the both sides of micro-fluidic chip pressure channel, is out of shape the time dependent waveform of impedance by low frequency corresponding in pressure channel process and high frequency two Frequency point pressure channel both sides for recording cell to be measured under suction function.

In the present embodiment, impedance information acquisition module is lock-in amplifier.In Fig. 4, (A) is under two Frequency points, when cell continues through pressure channel, and the time dependent curve of pressure channel both sides impedance magnitude; In Fig. 4, (B) is under two Frequency points, when cell continues through pressure channel, and the time dependent curve of pressure channel both sides impedance phase.

It should be noted that, in the present embodiment, low frequency be the condensance of cell membrane under this frequency much larger than frequency corresponding to the frequency of cell peripheral leakage resistance size, actual value greatly about 10kHz with lower frequency, representative value is 1kHz; High frequency is that the condensance of cell membrane under this frequency and cell peripheral leakage resistance can be comparable, and actual value is greatly about more than 10kHz, and representative value is 100kHz.

In addition, when cell to be measured is by pressure channel, image detection module is recorded to the image of cell tensile, and image procossing just can obtain being in the length L of the cell under extended state in pressure channel _cell? _elongation(as Suo Shi (C) in Fig. 4).

Data processing module, is connected with impedance information acquisition module with described image detection module, comprises: Young modulus obtains submodule, and the image for being taken by camera obtains the transient Displacements X that cell to be measured enters pressure channel _{instantaneous}with unstability displacement X _transitional, and then in conjunction with the diameter d of cell to be measured _cell? _diameter, the logical size W of compression _constriction? _channel, negative pressure numerical value P _aspirationcalculate the Young modulus of cell to be measured; Cell membrane ratio capacitance obtains submodule, for extract in the waveform that obtained by electric impedance analyzer do not have cell by time pressure channel two ends low-frequency impedance Z _l0(ω) with high-frequency resistance Z _h0(ω), have cell by time pressure channel two ends low-frequency impedance Z _l1(ω), high-frequency resistance Z _h1, and then calculate the film ratio capacitance of cell to be measured (ω).

Young modulus obtains submodule for utilizing the Young modulus of following formulae discovery cell to be measured:

Work as d _cell? _diameterduring < 14.0 μm:

$\frac{X_{\mathrm{ins}\tan \tan \mathrm{eous}}}{W_{\mathrm{constrictionchannel}}}=(222.8\&times;f_c^2-169.7\&times;f_c+39.7)\&times;\frac{P_{\mathrm{aspiration}}}{E_{\mathrm{Youn}{g}^{\&prime;}\mathrm{smdoulus}}}+(-21.5\&times;f_c^2+12.3\&times;f_c-1.8)---(1-1)$

$\frac{X_{\mathrm{transitional}}}{W_{\mathrm{constrictionchannel}}}=(237.4\&times;f_c^2-106.6\&times;f_c+7.2)\&times;\frac{P_{\mathrm{aspiration}}}{E_{{\mathrm{Young}}^{\&prime;}\mathrm{smdoulus}}}+(-8.5\&times;f_c^2+4.4\&times;f_c+0.5)---(1-2)$

As 16.0 μm of > d _cell? _diameterduring > 14.0 μm:

$\frac{X_{\mathrm{ins}\tan \tan \mathrm{eous}}}{W_{\mathrm{constrictionchannel}}}=(218.5\&times;f_c^2-137.4\&times;f_c+26.9)\&times;\frac{P_{\mathrm{aspiration}}}{E_{\mathrm{Youn}{g}^{\&prime;}\mathrm{smdoulus}}}+(-16.6\&times;f_c^2+9.8\&times;f_c-1.5)---(2-1)$

$\frac{X_{\mathrm{transitional}}}{W_{\mathrm{constrictionchannel}}}=(-213.5\&times;f_c^2+106.8\&times;f_c-17.8)\&times;\frac{P_{\mathrm{aspiration}}}{E_{{\mathrm{Young}}^{\&prime;}\mathrm{smdoulus}}}+(23.3\&times;f_c^2-10.2\&times;f_c+2.9)---(2-2)$

Work as d _cell? _diameterduring > 16.0 μm:

$\frac{X_{\mathrm{ins}\tan \tan \mathrm{eous}}}{W_{\mathrm{constrictionchannel}}}=(32.6\&times;f_c^2-30.2\&times;f_c+9.3)\&times;\frac{P_{\mathrm{aspiration}}}{E_{\mathrm{Youn}{g}^{\&prime;}\mathrm{smdoulus}}}+(-0.4\&times;f_c^2+1.2\&times;f_c-0.3)---(3-1)$

$\frac{X_{\mathrm{transitional}}}{W_{\mathrm{constrictionchannel}}}=(-108.4\&times;f_c^2+60.3\&times;f_c-11.2)\&times;\frac{P_{\mathrm{aspiration}}}{E_{{\mathrm{Young}}^{\&prime;}\mathrm{smdoulus}}}+(16.8\&times;f_c^2-8.9\&times;f_c+3.4)---(3-2)$

Wherein, d _cell? _dianeterfor the diameter of cell to be measured, W _constriction? _channelfor the size of foursquare pressure channel, P _aspirationfor the numerical value of negative pressure, X _{instantaneous}and X _transitionalcell to be measured enters transient Displacements and the unstability displacement of pressure channel respectively, f _cfor the friction factor of pressure channel wall.W _constriction? _channeland P _aspirationfor known quantity; d _cell? _diameter, X _{instantaneous}and X _transitionalfor the image obtained by image detection module is obtained through image procossing

It should be noted that, above-mentioned formula is the physical process entering pressure channel based on ABAQUS Mechanics Simulation software simulation cell, obtain cell front end instantaneous enter pressure channel displacement and the obtaining of relation of geometric parameter of the size of cell, Young modulus, pressure and pressure channel.Substitute into these formula by data experiment obtained, just can obtain the Young modulus of cell.

In the present embodiment, the version of the ABAQUS wherein used is 6.11, and mode cross section is the size W of foursquare pressure channel _constriction? _channel10 microns, cell division unit is CPE4RH, and division unit quantity is 5651, and cell physical characteristics is defined as super viscoelastic body.Obtain cell based on ABAQUS and enter the process of pressure channel as shown in (A) in Fig. 5-(C) with the rising of pressure, obtain transient Displacements X _{instantaneous}with unstability displacement X _transitionalwith relation (corresponding cell dia d as Suo Shi (D) in Fig. 5 of Young's Moduli _cell? _diameter15 microns), wherein, formula 1. ~ be 5. " unstability displacement/pressure channel width " and the linear fit formula of " sucking pressure/Young's Moduli ", formula 6. ~ be 10. " transient Displacements/pressure channel width " and the linear fit formula of " sucking pressure/Young's Moduli ", the computing formula of Young modulus can be converted to by these formula, thus obtain the Young modulus (as shown in Table 1) of individual cells.

Table one is for by the cell dia of the cell to be measured of pressure channel, transient Displacements, unstability displacement and utilize said method to calculate the Young modulus obtained.

Table one

Based on theoretical analysis, obtain cell membrane equivalent capacity, tenuigenin equivalent resistance, between cell and pressure channel wall the electrical parameter of leakage resistance and pressure channel self and the relation of impedance spectrum as follows:

(1) equivalent electrical model time acellular is the equivalent resistance R of cell culture fluid in pressure channel _mwith system parasitic electric capacity C _parasitic;

(2) cell is the cell membrane fractions equivalent capacity C vertical with pressure channel fluid flow direction by equivalent electrical model during pressure channel _membranewith represent cytoplasmic equivalent resistance R _cytoplasmseries connection, forms cell branch road, cell branch road and the leakage resistance R that can not fill pressure channel completely due to cell and be formed _leakin parallel; Use R _mreplace equivalent resistance R _m, R _mthe equivalent resistance of cell by cell culture fluid during pressure channel.

Fig. 6 is the equivalent-circuit model of pressure channel, wherein R _mand C _parasiticrepresent equivalent resistance and the system parasitic electric capacity of cell culture fluid in pressure channel respectively.C _membranerepresent the cell membrane fractions equivalent capacity vertical with pressure channel fluid flow direction, R _cytoplasmrepresent cytoplasmic equivalent resistance, R _leakthe leakage resistance that representative can not be filled pressure channel completely due to cell and be formed, R _mrepresent the equivalent resistance of cell culture fluid in pressure channel.

In Fig. 6, (A) is for when there being cell by pressure channel, impedance operator measuring system schematic diagram, the figure shows in whole impedance measurement loop, the equivalent electrical circuit that when (B) represents acellular in the capacitance resistance existed and their distribution Fig. 6, high frequency (as 100kHz) electric current is corresponding, the equivalent electrical circuit that when (C) indicates cell in Fig. 6, high frequency (as 100kHz) electric current is corresponding, the equivalent electrical circuit that when (D) represents acellular in Fig. 6, low frequency (as 1kHz) electric current is corresponding, the equivalent electrical circuit that when (E) indicates cell in Fig. 6, low frequency (as 1kHz) electric current is corresponding.

By equivalent electrical circuit, obtain following equivalent electrical formula:

Wherein when do not have cell by time, equiva lent impedance can be expressed as:

$Z_0\left(\mathrm{\&omega;}\right)=R_m//\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}}---(4-1)$

Wherein ω is that 2 π are multiplied by frequency values, and equation (4-1) is circuit description, wherein " // " indication circuit parallel connection (all same below).

And be finally expressed as:

$Z_0\left(\mathrm{\&omega;}\right)=\frac{R_m}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}{R}_m+1}---(4-2)$

Simultaneously at low frequency, the impedance of stray capacitance much larger than solution resistance impedance, so think that the impedance measured is solution resistance impedance.

When have cell by time, equiva lent impedance can be expressed as:

$Z_{d1}\left(\mathrm{\&omega;}\right)=\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}}//(R_m^{\&prime;}+(R_{\mathrm{leak}}//(2\&times;\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{membrane}}}+R_{\mathrm{cytoplasm}})))---(4-3)$

Wherein " // " indication circuit is in parallel, and "+" indication circuit is connected.

The impedance of cell electronic circuit is:

$Z_{\mathrm{cell}}\left(\mathrm{\&omega;}\right)=2\&times;\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{membrane}}}+R_{\mathrm{cytoplasm}}---(4-4)$

Stray capacitance impedance is:

$Z_{\mathrm{parasitic}}\left(\mathrm{\&omega;}\right)=\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}}---(4-5)$

When have cell by time, equiva lent impedance is:

$Z_1\left(\mathrm{\&omega;}\right)=\frac{Z_{\mathrm{parasitic}}\&times;(R_m^{\&prime;}\&times;(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\&times;R_{\mathrm{leak}}))}{(Z_{\mathrm{parasitic}}+R_m^{\&prime;})\&times;(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\&times;R_{\mathrm{leak}}}---(4-6)$

On the basis that above-mentioned theory is analyzed, cell membrane ratio capacitance obtains submodule, for the cell membrane ratio capacitance C utilizing following complex number equation group to calculate cell to be measured _specific? _membrane:

$Z_{\mathrm{LF}0}\left(\mathrm{\&omega;}\right)=R_m=\&Integral;\frac{d{L}_{\mathrm{channel}}}{S_{\mathrm{Channel}}\&times;{\mathrm{\&sigma;}}_{\mathrm{RPMI}}}---(5-1)$

$Z_{\mathrm{HF}0}\left(\mathrm{\&omega;}\right)=\frac{R_m}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}{R}_m+1}---(5-2)$

$Z_{\mathrm{LF}1}\left(\mathrm{\&omega;}\right)=R_m-\frac{L_{\mathrm{cellelongation}}}{S_{\mathrm{constrictionChannel}}\&times;{\mathrm{\&sigma;}}_{\mathrm{RPMI}}}---(5-3)$

$Z_{\mathrm{HF}1}\left(\mathrm{\&omega;}\right)=\frac{Z_{\mathrm{parasitic}}\&times;(R_m^{\&prime;}\&times;(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\&times;R_{\mathrm{leak}}))}{(Z_{\mathrm{parasitic}}+R_m^{\&prime;})\&times;(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\&times;R_{\mathrm{leak}}}---(5-4)$

$Z_{\mathrm{cell}}\left(\mathrm{\&omega;}\right)=2\&times;\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{membrane}}}+R_{\mathrm{cytoplasm}}---(5-5)$

$Z_{\mathrm{parasitic}}\left(\mathrm{\&omega;}\right)=\frac{1}{\mathrm{j\&omega;}{C}_{\mathrm{parasitic}}}---(5-7)$

$C_{\mathrm{specificmembrane}}=\frac{C_{\mathrm{membrane}}}{S_{\mathrm{constrictionChannel}}}---(5-8)$

Wherein, S _channelfor whole passage comprises the cross-sectional area at integration place of pressure channel and introduction passage each several part, S _constriction? _channelfor the cross-sectional area of pressure channel, be W _constriction? _channelsquare, be known quantity.Z _lF0(ω), Z _hF0(ω), Z _lF1(ω), Z _hF1(ω) be respectively impedance information module acquires to there is no a cell time and have cell by pressure channel time, the resistance value of corresponding low frequency and high-frequency compression passage both sides, wherein subscript LF represents low frequency, and HF represents high frequency, 0 represents do not have cell, and 1 indicates cell.L _cell? _elongationrepresent cell elongation length, after photographing by image capture module the image that cell extends in pressure channel, obtained by image procossing.σ _rPMIrepresent cell culture fluid conductivity, C _parasiticfor system parasitic electric capacity, R _cytoplasmfor tenuigenin equivalent resistance, C _membranefor cell membrane equivalent capacity, being unknown quantity, can obtaining by solving complex number equation group.

Table two is for by the parameter of the cell to be measured of pressure channel and utilize above-mentioned parameter to calculate the cell membrane ratio capacitance obtained.

Table two

So far, by reference to the accompanying drawings the present embodiment has been described in detail.Describe according to above, the system that those skilled in the art should characterize unicellular Young modulus and cell membrane ratio capacitance to the present invention has simultaneously had clearly to be familiar with.

In addition, the above-mentioned definition to each element and method is not limited in various concrete structures, shape or the mode mentioned in embodiment, and those of ordinary skill in the art can change simply it or replace, such as:

(1) except lock-in amplifier, impedance information acquisition module can also use the impedance measuring equipments such as electric impedance analyzer to replace;

(2) except 1kHz, low frequency can use the lower frequencies such as 2kHz (namely under this frequency cell membrane capacitance impedance much larger than frequency corresponding to cell peripheral leakage resistance impedance) to replace; Same except 100kHz, high frequency can adopt the upper frequencies such as 200kHz (namely under this frequency cell membrane capacitance impedance much larger than frequency corresponding to cell peripheral leakage resistance impedance) to replace.

In sum, the present invention proposes a kind of system simultaneously characterizing unicellular Young modulus and cell membrane ratio capacitance, the high flux of the Young modulus and cell membrane ratio capacitance that realize the intrinsic mechanics of cell and electrology characteristic parameter and cell gathers simultaneously, sign for cell biological physical characteristics provides reliable method and access, can be the disease that anaemia, tumour etc. exist the corresponding change of cell biological physical characteristics and provides new detection means and the new cell characteristics mark without the need to mark.

Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (11)

1. characterize a system for unicellular Young modulus and cell membrane ratio capacitance simultaneously, it is characterized in that, comprising:

Micro-fluidic chip, comprising:

Transparent substrates,

Supporting body, is formed at described transparent substrates front; And

Pressure channel, be formed at described supporting body inner, its lower surface is the upper surface of described transparent substrates, and for passing through for cell compression to be measured, the cross-sectional area of this pressure channel is less than the cross-sectional area of described cell to be measured;

Image detection module, for taking cell to be measured process by described pressure channel under suction function from the lower surface of described transparent substrates;

Impedance information acquisition module, its two potential electrode stretches into the both sides of described pressure channel, for recording compressed passage both sides, the time dependent waveform of impedance of corresponding low frequency and high frequency two Frequency points;

Data processing module, is connected with impedance information acquisition module impedance information acquisition module with described image detection module, comprises:

Young modulus obtains submodule, and the image for being taken by camera obtains the transient Displacements X that cell to be measured enters pressure channel _{instantaneous}with unstability displacement X _transitional, and then in conjunction with the diameter d of cell to be measured _cell? _diameter, the logical size W of compression _constriction? _channel, negative pressure numerical value P _aspirationcalculate the Young modulus of cell to be measured;

Cell membrane ratio capacitance obtains submodule, obtains for the image taken by camera the length L being in the cell under extended state in pressure channel _cell? _elongation, extract in the waveform obtained by electric impedance analyzer do not have cell by time pressure channel two ends low-frequency impedance Z _l0(ω) with high-frequency resistance Z _h0(ω), have cell by time pressure channel two ends low-frequency impedance Z _l1(ω), high-frequency resistance Z _h1, and then calculate the film ratio capacitance of cell to be measured (ω).

2. system according to claim 1, is characterized in that:

The material of described transparent substrates is glass or dimethyl silicone polymer;

Described supporting body is that integrated injection molding is formed on the transparent substrate, and its material is polydimethylsiloxanes material, organic glass or SU8 material.

3. system according to claim 2, is characterized in that, described micro-fluidic chip is prepared in the following ways:

Steps A, applies one deck SU-85 photoresist in microslide Rotating with Uniform;

Step B, to the described SU-85 photoresist front baking on microslide, exposure; Do not develop, rear baking, thus form pressure channel formpiston;

Step C, on described exposed SU-85 photoresist, Rotating with Uniform applies one deck SU-825 photoresist again;

Step D, carries out front baking to described SU-825 photoresist, after pattern alignment figure in lithography mask version and described SU-85 are exposed, exposes described SU-825 photoresist;

Step e, to drying after the SU-825 photoresist after described exposure, developing;

Step F, carries out perpendicular film to described SU-85 and SU-825 photoresist, forms the formpiston comprising the two-layer ledge structure of cell introduction passage and pressure channel;

Step G, then at performed polymer and the hardening agent of the liquid dimethyl silicone polymer of described formpiston upper;

Step H, obtains the performed polymer of the liquid dimethyl silicone polymer be cast on described formpiston and hardening agent curing and demolding the dimethyl silicone polymer block that there is microfluidic channel on surface, i.e. the carrier section of chip;

Step I, punches described dimethyl silicone polymer block, makes it through in introduction passage end positions upper and lower surface; And

Step J, by the dimethyl silicone polymer block after described punching and glass substrate sealing-in, wherein dimethyl silicone polymer block has one side and the glass contact of microfluidic channel, completes the making of micro-fluidic chip.

4. system according to claim 1, is characterized in that, the xsect of described pressure channel is square, and its cross-sectional area is the 40%-90% that cell cross section to be measured amasss.

5. system according to claim 1, is characterized in that, described negative pressure is between 100Pa to 10kPa.

6. system according to claim 1, is characterized in that, described image detection module comprises:

Biological inverted microscope, its image-forming component aims at described pressure channel from the back side of described transparent substrates, can the image of camera-shot for being enlarged into by described cell to be measured;

Camera, aims at biological inverted microscope and arranges, for taking cell to be measured process by described pressure channel under suction function by described biological inverted microscope.

7. system according to claim 6, is characterized in that, described microscopical enlargement factor is 400 times, and the sweep velocity of camera is 200 frames/second.

8. system according to claim 1, is characterized in that, described impedance information acquisition module is lock-in amplifier or electric impedance analyzer, and described low frequency is the frequency of below 10kHz, and described high frequency is the frequency of more than 10kHz.

9. system according to claim 8, is characterized in that, described low frequency is 1kHz, and described high frequency is 100kHz.

10. system according to any one of claim 1 to 9, is characterized in that, described Young modulus obtains the Young modulus that submodule utilizes following formulae discovery cell to be measured:

Work as d _cell? _diameterduring < 14.0 μm:

As 16.0 μm of > d _cell? _diameterduring > 14.0 μm:

Or, work as d _cell? _diameterduring > 16.0 μm:

Wherein, d _cell? _diameterfor the diameter of cell to be measured, W _constriction? _channelfor the length of side of foursquare pressure channel, P _aspirationfor the numerical value of negative pressure, X _{instantaneous}and X _transitionalcell to be measured enters transient Displacements and the unstability displacement of pressure channel respectively, f _cfor the friction factor of the pressure channel wall of the unknown, W _constriction? _channeland P _aspirationfor known quantity; d _cell? _diameter, X _{instantaneous}and X _transitionalfor the image obtained by image detection module is obtained through image procossing.

11. systems according to any one of claim 1 to 9, is characterized in that, described cell membrane ratio capacitance obtains the film ratio capacitance that submodule is used for calculating according to following complex number equation group cell to be measured:

Wherein, C _membranefor cell membrane equivalent capacity, S _channelfor whole passage comprises the cross-sectional area at integration place of pressure channel and introduction passage each several part; S _constriction? _channelfor the cross-sectional area of pressure channel; Z _lF0(ω), Z _hF0(ω), Z _lF1(ω), Z _hF1(ω) be respectively that impedance information acquisition module collects there is no a cell time and have cell by pressure channel time, the resistance value of pressure channel both sides when corresponding low frequency and high frequency, wherein subscript LF represents low frequency, and HF represents high frequency, 0 represents do not have cell, and 1 indicates cell; L _cell? _elongationrepresent cell elongation length; σ _rPMIrepresent cell culture fluid conductivity, C _parasiticfor system parasitic electric capacity, R _cytoplasmfor tenuigenin equivalent resistance.