CN104251810B - Characterize unicellular Young's modulus and the cell membrane system than electric capacity simultaneously - Google Patents

Abstract

The invention provides a kind of based on characterizing unicellular Young's modulus and the cell membrane system than electric capacity while microflow control technique.Cell is equivalent to isotropic super viscoelastic body by this system, obtain displacement and the relation of the geometric parameter of the size of cell, Young's modulus, pressure and pressure channel of the instantaneous entrance in cell front end pressure channel based on ABAQUS Mechanics Simulation, be calculated the Young's modulus of cell；In addition, the cell equivalent electrical model by pressure channel is also proposed, obtain the electrical parameter of cell membrane capacitance, Cytoplasm resistance, cell and pressure channel wall ohmic leakage and pressure channel self and the relation of impedance spectrum, cause the change of impedance to be converted into cell membrane by pressure channel in cell and compare electric capacity, thus be calculated single celled cell membrane than electric capacity, it is achieved thereby that unicellular Young's modulus and cell membrane are than the synchro measure of electric capacity.

Description

Characterize unicellular Young's modulus and the cell membrane system than electric capacity simultaneously

Technical field

The present invention relates to bio information detection field, particularly relate to one and characterize unicellular Young mould simultaneously Amount and cell membrane are than the system of electric capacity.

Background technology

Cell is as the ultimate unit of vital movement, containing various biomolecule, phase interaction between them With, collectively form a busy and orderly system.The mechanics parameters of cell i.e. Young's modulus, Determined by cytoskeleton；The electrology characteristic parameter i.e. cell membrane of cell is than electric capacity (cell membrane unit are Electric capacity), be made up of with memebrane protein phospholipid bilayer.Cytoskeleton and cell membrane are as cell Critical function unit, participates in the physiological function that cell proliferation, division etc. are important, close with cell state Relevant.Although traditional characterization technique such as atomic force microscope, patch-clamp etc. can preliminary characterization cells Young's modulus and cell membrane than electric capacity, but above-mentioned technology for detection flux is low and cannot characterize simultaneously The mechanics of cell and electrology characteristic, limit the sign of cell biological physical characteristic.

Micro-fluidic chip is referred to as " chip lab ", be by semiconductor integration technology make new Type solid-state components, can carry out complexity, operate accurately micro fluid.Spy due to micro-fluidic chip Levy size comparable with cell size, be suitable to single celled capture and characteristic present, be the most tentatively used for Gather the mechanics/electrology characteristic of cell simultaneously.

The Prof.Fujii seminar of Tokyo Univ Japan in 2006 proposes cantilever based on microflow control technique Beam sequence capturing individual cells, uses cantilever beam electrode to characterize the electrology characteristic of captured cell, simultaneously The mechanical characteristic of individual cells is characterized according to the deformation extent causing cantilever beam during cell capture.Should Seminar uses this method successfully to distinguish normal and ill erythrocyte.

During realizing the present invention, it is found by the applicant that existing cyto-mechanics/electrology characteristic high flux The method synchronizing to characterize has following defects that and i.e. can only characterize some biological things depending on cell size Reason characterisitic parameter is as entered the time of pressure channel, cantilever beam degree of deformation, impedance spectrum etc., it is impossible to table Levy the intrinsic mechanics of cell/electrology characteristic parameter i.e. Young's modulus of cell and cell membrane compares electric capacity.

Summary of the invention

(1) to solve the technical problem that

In view of above-mentioned technical problem, the invention provides a kind of single based on characterizing while microflow control technique Young's Moduli and cell membrane are than the system of electric capacity, to realize individual cells Young's modulus and cell membrane Synchronize to characterize than the high flux of electric capacity.

(2) technical scheme

The invention provides and a kind of characterize unicellular Young's modulus and the cell membrane system than electric capacity simultaneously. This characterizes unicellular Young's modulus simultaneously and cell membrane includes than the system of electric capacity: micro-fluidic chip, figure As detection module, impedance information acquisition module and data processing module.

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

Image detection module, makees in negative pressure for shooting cell to be measured from the lower surface of described transparent substrates By the lower process by described pressure channel.

Impedance information acquisition module, its two measurements electrode stretches into the both sides of described pressure channel, is used for remembering Record pressure channel both sides, corresponding low frequency and the time dependent waveform of impedance of two Frequency points of high frequency.

Data processing module, adopts with described image detection module and impedance information acquisition module impedance information Collection module is connected, including: Young's modulus obtains submodule, obtains for the image shot by photographic head Obtain cell to be measured and enter transient Displacements X of pressure channel_{instantaneous}With unstability displacement X_transitional, enter And combine the diameter d of cell to be measured_{cell diameter}, logical size W of compression_{constriction channel}, negative pressure numerical value P_aspirationCalculate the Young's modulus of cell to be measured；Cell membrane obtains submodule than electric capacity, for by taking the photograph As the image of head shooting obtains length L of the cell being under extended state in pressure channel_{cell elongation}, The waveform obtained by electric impedance analyzer extracts the low frequency resistance at pressure channel two ends when cell passes through Anti-Z_L0(ω) with high-frequency resistance Z_H0(ω) the low-frequency impedance Z at pressure channel two ends when, having cell to pass through_L1(ω), High-frequency resistance Z_H1, and then calculate the film of cell to be measured and compare electric capacity (ω).

(3) beneficial effect

From technique scheme it can be seen that the present invention characterizes unicellular Young's modulus and cell membrane simultaneously Have the advantages that than the system of electric capacity

(1) cell is equivalent to isotropic super viscoelastic body, based on ABAQUS Mechanics Simulation Obtain the displacement of the instantaneous entrance in cell front end pressure channel and the size of cell, Young's modulus, pressure and The relation of the geometric parameter of pressure channel, is calculated the Young's modulus of cell；

(2) propose the cell equivalent electrical model by pressure channel, obtain cell membrane capacitance, thin Kytoplasm resistance, cell and pressure channel wall ohmic leakage and the electrical parameter of pressure channel self and impedance The relation of frequency spectrum, causes the change of impedance to be converted into cell membrane than electric capacity by pressure channel in cell, Thus be calculated single celled cell membrane and compare electric capacity；

(3) present invention proposes the method simultaneously characterizing the Young's modulus of cell and cell membrane than electric capacity, Realize the intrinsic mechanics of cell and the electrology characteristic parameter i.e. Young's modulus of cell and cell membrane compares electric capacity High flux gather simultaneously, the sign for cell biological physical characteristic provides reliable method and approach；

(4) micro-fluidic chip used chooses the low cost material such as microscope slide and polydimethylsiloxane Material is processed, based on fine machining method, have low cost, can mass manufacture, disposable etc. Feature；

Summary advantage, it is corresponding that the present invention can be that anemia, tumor etc. exist cell biological physical characteristic The disease changed provides new detection means and the new cell characteristics mark without labelling.

Accompanying drawing explanation

Fig. 1 is to characterize unicellular Young's modulus and cell membrane according to the embodiment of the present invention than electric capacity simultaneously The structural representation of system.

Fig. 2 is the flow chart 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 pressure channel under the effect of the pressure Picture and measurement obtain cell front end and are compressed the displacement of passage and the relation of time；

Fig. 4 be application example of the present invention gather cell by pressure channel cause the change of double frequency impedance with And the image of certain cell being positioned in pressure channel；

The cell that Fig. 5 is the present invention to be obtained based on computer simulation enter the simulation result of pressure channel with And the relation of the Young's modulus of the transient Displacements of cell, unstability displacement and cell；

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

Detailed description of the invention

For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with concrete real Execute example, and referring to the drawings, the present invention is described in more detail.It should be noted that at accompanying drawing or During description describes, similar or identical part all uses identical figure number.Accompanying drawing does not illustrates or retouches The implementation stated, for form known to a person of ordinary skill in the art in art.Although it addition, The demonstration of the parameter comprising particular value can be provided herein, it is to be understood that parameter is equal to accordingly without definite Value, but can be similar to be worth accordingly in acceptable error margin or design constraint.Embodiment In the direction term mentioned, such as " on ", D score, "front", "rear", "left", "right" etc., only It it is the direction with reference to accompanying drawing.Therefore, the direction term of use is used to illustrate not for limiting this Bright protection domain.

The present invention provides one to realize the intrinsic mechanical characteristic of individual cells (i.e. Young's modulus) and electricity is special Property (i.e. cell membrane is than electric capacity) high flux synchronize characterize system.

In one exemplary embodiment of the present invention, it is provided that one characterizes unicellular Young mould simultaneously Amount and cell membrane are than the system of electric capacity.Fig. 1 for characterizing unicellular Young simultaneously according to the embodiment of the present invention Modulus and cell membrane are than the structural representation of the system of electric capacity.Refer to Fig. 1, the present embodiment table simultaneously Levy unicellular Young's modulus and cell membrane includes than the system of electric capacity: micro-fluidic chip, image detection mould Block, impedance information acquisition module and data processing module.

Individually below the present embodiment is characterized simultaneously unicellular Young's modulus and cell membrane than electric capacity system Each ingredient of system is described in detail.

Refer to Fig. 1, micro-fluidic chip, including: transparent glass substrate；Supporting body, its material For polydimethylsiloxane, it is formed at described transparent substrates front；And be formed at inside supporting body The pressure channel passed through for cell compression to be detected, the cross-sectional area of this pressure channel is less than cell Cross-sectional area.Cell to be measured deforms under the effect of negative pressure and squeezes through pressure channel, effectively stops electric field line, Reduce leakage current.

In the present embodiment, in addition to a glass substrate, it is also possible to employing polydimethylsiloxane etc. are transparent Plastic material is used as substrate；In addition to polydimethylsiloxane, it is also possible to use lucite, The transparent plastic material such as SU8 is molded the above-mentioned supporting body of formation.

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

In addition to pressure channel, above-mentioned supporting body is also formed with such as lower part: two sections of cells introduce logical Road, is respectively formed in the left and right sides of pressure channel on supporting body, and its cross section is similarly square, Size is more than the cross sectional dimensions of described pressure channel；Sample injection port, is extended by supporting body upper surface To left side cell introduction passage, for injecting the sample comprising cell to be measured；Sample suction outlet, by holding Carrier upper surface extends to right side cell introduction passage, and its upper end is by airtight hose connection to air pressure control Device processed, for being provided negative pressure that cell is drawn through pressure channel by gas pressure regulator to be measured carefully Born of the same parents deform by described pressure channel.

In the present embodiment, the diameter d of cell to be measured_{cell-diameter}Being about 15.6 μm, accordingly, compression is logical The cross section in road is square, its length of side W_{constriction channel}It is 10 μm, the sample at pressure channel two ends The height in product pond is 45 μm.Via experiment, determine that the negative pressure drawing cell is 500 handkerchiefs.

Micro-fluidic chip is double-deck pressure channel based on polydimethylsiloxane, uses microfabrication work Skill makes.Fig. 2 is the flow chart of micro-flow control chip preparation method in system described in Fig. 1.Such as Fig. 2 institute Showing, the preparation process of this micro-fluidic chip is as follows:

Step S202, microscope slide (75 millimeters long, 25 mm wides and 1 millimeters thick) is in acetone, second Alcohol and deionized water clean, dries (150 DEG C, 30 minutes), apply one in microscope slide Rotating with Uniform Layer SU-85 (1500RPM, 35 seconds), as shown in (A) in Fig. 2；

Step S204, on microscope slide SU-85 photoresist front baking (65 DEG C, 2 minutes；95 DEG C, 5 minutes), expose (60mW/cm²), as shown in (B) in Fig. 2, do not develop, after bake (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 layer again SU-825 photoresist (2000RPM, 35 seconds), as shown in (C) in Fig. 2；

Step S208, above-mentioned SU-825 photoresist is carried out front baking (65 DEG C, 3 minutes；95 DEG C, 7 minutes), after the image alignment of the image in photo mask board and SU-85 exposure, to SU-525 Photoresist is exposed (75mW/cm²), as shown in (D) in Fig. 2；

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

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

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

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

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

Step S220, it is achieved itself and glass substrate sealing-in, completes micro-fluidic chip, such as (I) in Fig. 2 Shown in.

Above-mentioned preparation technology is for being engaged in the technical staff in micro-fluidic chip field, by above-mentioned preparation The description of process, and combine accompanying drawing, the structure understanding this micro-fluidic chip that can will be apparent from, herein Repeat no more.

Image detection module is for shooting cell to be measured under suction function by described pressure channel Process.Refer to Fig. 1, this image detection module includes: image-forming component is from the back of the body of transparent glass substrate In the face of biological inverted microscope and the photographic head of the biological inverted microscope of alignment of accurate described pressure channel, Wherein, described biological inverted microscope is for small channel image and cell image etc. are converted to can The image size of camera-shot；Photographic head is for bearing by biological inverted microscope record cell Image information during deforming by described pressure channel under pressure effect.

In the present embodiment, microscopical amplification is 400 times, and the scanning speed of photographic head is 200 Frame/second.It is 20 collection points per second for gathering the sample frequency of the electric impedance analyzer of impedance operator, Measuring frequency is 1kHz and 100kHz.

The present embodiment in use, uses negative pressure that cell is drawn through pressure channel continuously, and image is examined Survey module real time record cell its front end under suction function gradually extend into and pass through pressure channel Physical process (as in Fig. 3 scheme A～figure D shown in).By (A) in Fig. 3～(D), can obtain During gradually extending through pressure channel to cell, front end changes over relative to the displacement of entrance Curve, as shown in (E) in Fig. 3.Just can show that by this curve cell to be measured enters compression logical The instantaneous displacement in road, unstability displacement.

Wherein, cell to be measured is under suction function, and moment is inhaled in pressure channel, produces one immediately Fixed displacement, the immediate movement of this displacement cell the most to be measured.Owing to cell belongs to viscoelastic body material, In the moment by External Force Acting, elasticity plays a major role, and produces obvious deformation, i.e. immediate movement, Namely cell is in the displacement in zero positive moment.Due to the reason of pressure channel shape, cell is entering pressure During contracting passage, when entering into certain displacement, the power wink that suffered resistance and negative pressure are applied Between uneven, produce the biggest acceleration change, in this Place cell front end relative to pressure channel entrance Displacement be referred to as unstability displacement.By above-mentioned instantaneous displacement and unstability displacement, it is possible to combine thin The information such as born of the same parents' diameter, the pressure channel length of side calculate the Young module of cell to be measured, will in detail below Describe.

Impedance information acquisition module, its two measurements electrode stretches into the both sides of micro-fluidic chip pressure channel, For recording corresponding low frequency and height during cell to be measured deforms by pressure channel under suction function Frequently the time dependent waveform of impedance of two Frequency point pressure channel both sides.

In the present embodiment, impedance information acquisition module is lock-in amplifier.In Fig. 4, (A) is two Under Frequency point, when cell continues through pressure channel, pressure channel both sides impedance magnitude changes over Curve；Under in Fig. 4, (B) is two Frequency points, when cell continues through pressure channel, compression The passage both sides time dependent curve of impedance phase.

It should be noted that in the present embodiment, low frequency is that under this frequency, the condensance of cell membrane is long-range In the frequency that the frequency of cell peripheral ohmic leakage size is corresponding, actual value about in 10kHz frequencies below, Representative value is 1kHz；High frequency is that under this frequency, the condensance of cell membrane and cell peripheral ohmic leakage can With comparable, actual value is about at more than 10kHz, and representative value is 100kHz.

Additionally, when cell to be measured is by pressure channel, image detection module recorded cell tensile Image, image procossing can be obtained by being in the length of the cell under extended state in pressure channel L_{cell elongation}(as shown in (C) in Fig. 4).

Data processing module, is connected with described image detection module and impedance information acquisition module, bag Including: Young's modulus obtains submodule, the image for being shot by photographic head obtains cell to be measured and enters pressure Transient Displacements X of contracting passage_{instantaneous}With unstability displacement X_transitional, and then combine cell to be measured Diameter d_{cell diameter}, logical size W of compression_{constriction channel}, negative pressure numerical value P_aspirationCalculate to be measured carefully The Young's modulus of born of the same parents；Cell membrane obtains submodule than electric capacity, for the waveform obtained by electric impedance analyzer The low-frequency impedance Z at pressure channel two ends when middle extraction does not has cell to pass through_L0(ω) with high-frequency resistance Z_H0(ω), The low-frequency impedance Z at pressure channel two ends when having cell to pass through_L1(ω), high-frequency resistance Z_H1(ω), so calculate The film of cell to be measured compares electric capacity.

Young's modulus obtains submodule for utilizing the Young's modulus of equation below calculating cell to be measured:

Work as d_{cell diameter}During ＜ 14.0 μm:

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

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

As 16.0 μm ＞ d_{cell diameter}During ＞ 14.0 μm:

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

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

Work as d_{cell diameter}During ＞ 16.0 μm:

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

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

Wherein, d_{cell dianeter}For the diameter of cell to be measured, W_{constriction channel}Lead to for foursquare compression The size in road, P_aspirationFor the numerical value of negative pressure, X_{instantaneous}And X_transitionalCell the most to be measured enters Enter transient Displacements and unstability displacement, the f of pressure channel_cCoefficient of friction for pressure channel wall.W_{constriction channel}And P_aspirationFor known quantity；d_{cell diameter}、X_{instantaneous}And X_transitionalFor being examined by image The image surveying module acquisition obtains through image procossing

It should be noted that above-mentioned formula is to enter based on ABAQUS Mechanics Simulation software analog cell Enter the physical process of pressure channel, obtain displacement and the cell of the instantaneous entrance in cell front end pressure channel Obtaining of the relation of the geometric parameter of size, Young's modulus, pressure and pressure channel.By by real The data testing acquisition substitute into these formula, it is possible to obtain the Young's modulus of cell.

In the present embodiment, the version of the ABAQUS wherein used is 6.11, and mode cross section is square Size W of pressure channel_{constriction channel}10 microns, cell division unit is CPE4RH, draws Subdivision quantity is 5651, and cell physical characteristic is defined as super viscoelastic body.Obtain based on ABAQUS Enter in the process such as Fig. 5 of pressure channel shown in (A)-(C) to cell with raising of pressure, obtain transient state Displacement X_{instantaneous}With unstability displacement X_transitionalWith (D) institute in the relation such as Fig. 5 of Young's Moduli Show (corresponding cell dia d_{cell diameter}15 microns), wherein, 1.～5. formula is " unstability displacement/pressure Contracting channel width " with the linear fit formula of " suck pressure/Young's Moduli ", 6.～10. formula Linear fit for " transient Displacements/pressure channel width " with " sucking pressure/Young's Moduli " Formula, can be converted to the computing formula of Young's modulus, thus obtain individual cells by these formula Young's modulus (as shown in Table 1).

Table one be by the cell dia of cell to be measured of pressure channel, transient Displacements, unstability displacement with And utilize said method to calculate the Young's modulus obtained.

Table one

Based on theory analysis, obtain cell membrane equivalent capacity, Cytoplasm equivalent resistance, cell and compression Between conduit wall, the electrical parameter of ohmic leakage and pressure channel self and the relation of impedance spectrum are as follows:

(1) equivalent electrical model time acellular is the equivalent resistance of cell culture fluid in pressure channel R_mWith system parasitic electric capacity C_parasitic；

(2) cell by equivalent electrical model during pressure channel is and pressure channel fluid flowing side To vertical cell membrane fractions equivalent capacity C_membraneWith represent cytoplasmic equivalent resistance R_cytoplasm Series connection, forms cell branch road, and cell branch road is formed with owing to cell can not be filled up completely with pressure channel Ohmic leakage R_leakIn parallel；Use R_mReplace equivalent resistance R_m, R_mIt is thin to be that cell passes through during pressure channel The equivalent resistance of born of the same parents' culture fluid.

Fig. 6 is the equivalent-circuit model of pressure channel, wherein R_mAnd C_parasiticRepresent that compression is logical respectively The equivalent resistance of cell culture fluid and system parasitic electric capacity in road.C_membraneRepresent and pressure channel stream The cell membrane fractions equivalent capacity that body flow direction is vertical, R_cytoplasmRepresent cytoplasmic equivalent resistance, R_leakRepresent the ohmic leakage formed owing to cell can not be filled up completely with pressure channel, R_mRepresent compression logical The equivalent resistance of cell culture fluid in road.

In Fig. 6, (A) is for when there being cell to pass through pressure channel, and impedance operator measures systematic schematic diagram, The figure shows in whole impedance measurement loop, the capacitance resistance existed and their distribution Fig. 6 In (B) equivalent circuit that high frequency (such as 100kHz) electric current is corresponding when representing acellular, in Fig. 6 (C) The equivalent circuit that when indicating cell, high frequency (such as 100kHz) electric current is corresponding, in Fig. 6 (D) indicate without The equivalent circuit that during cell, low frequency (such as 1kHz) electric current is corresponding, in Fig. 6, (E) indicates low during cell Frequently the equivalent circuit that electric current is corresponding (such as 1kHz).

By equivalent circuit, obtain following equivalent electrical formula:

Wherein when not having cell to pass through, equiva lent impedance can be expressed as:

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

Wherein ω is that 2 π are multiplied by frequency values, and equation (4-1) is that circuit describes, wherein " // " represent Circuit in parallel (the most same).

And be ultimately expressed as:

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

The most at low frequency, the impedance of parasitic capacitance is much larger than solution resistance impedance, it is believed that measure To impedance be solution resistance impedance.

When there being cell to pass through, equiva lent impedance can be expressed as:

$Z_{d1}\left(\mathrm{\ω}\right)=\frac{1}{\mathrm{j\ω}{C}_{\mathrm{parasitic}}}//(R_m^{\′}+(R_{\mathrm{leak}}//(2\×\frac{1}{\mathrm{j\ω}{C}_{\mathrm{membrane}}}+R_{\mathrm{cytoplasm}})))---(4-3)$

Wherein " // " indication circuit parallel connection, "+" indication circuit series connection.

Cell electronic circuit impedance is:

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

Parasitic capacitance impedance is:

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

When there being cell to pass through, equiva lent impedance is:

$Z_1\left(\mathrm{\ω}\right)=\frac{Z_{\mathrm{parasitic}}\×(R_m^{\′}\×(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\×R_{\mathrm{leak}}))}{(Z_{\mathrm{parasitic}}+R_m^{\′})\×(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\×R_{\mathrm{leak}}}---(4-6)$

On the basis of above-mentioned theory is analyzed, cell membrane obtains submodule than electric capacity, is used for utilizing as follows Complex number equation group calculates the cell membrane of cell to be measured than electric capacity C_{specific membrane}:

$Z_{\mathrm{LF}0}\left(\mathrm{\ω}\right)=R_m=\∫\frac{d{L}_{\mathrm{channel}}}{S_{\mathrm{Channel}}\×{\mathrm{\σ}}_{\mathrm{RPMI}}}---(5-1)$

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

$Z_{\mathrm{LF}1}\left(\mathrm{\ω}\right)=R_m-\frac{L_{\mathrm{cellelongation}}}{S_{\mathrm{constrictionChannel}}\×{\mathrm{\σ}}_{\mathrm{RPMI}}}---(5-3)$

$Z_{\mathrm{HF}1}\left(\mathrm{\ω}\right)=\frac{Z_{\mathrm{parasitic}}\×(R_m^{\′}\×(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\×R_{\mathrm{leak}}))}{(Z_{\mathrm{parasitic}}+R_m^{\′})\×(Z_{\mathrm{cell}}+R_{\mathrm{leak}})+Z_{\mathrm{cell}}\×R_{\mathrm{leak}}}---(5-4)$

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

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

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

Wherein, S_ChannelFor whole passage comprise pressure channel and introduction passage each several part at integration Cross-sectional area, S_{constriction Channel}For the cross-sectional area of pressure channel, it is W_{constriction channel} Square, for known quantity.Z_LF0(ω)、Z_HF0(ω)、Z_LF1(ω)、Z_HF1(ω) it is respectively impedance information module to adopt Collect to when there is no a cell and when having cell to pass through pressure channel, corresponding low frequency and high-frequency compression passage two The resistance value of side, wherein subscript LF represents that low frequency, HF represent high frequency, and 0 represents do not have cell, 1 Indicate cell.L_{cell elongation}Represent cell elongation length, photographed carefully by image capture module After the image that born of the same parents extend in pressure channel, obtained by image procossing.σ_RPMIRepresent cell culture fluid Electrical conductivity, C_parasiticFor system parasitic electric capacity, R_cytoplasmFor Cytoplasm equivalent resistance, C_membraneFor Cell membrane equivalent capacity, is unknown quantity, can obtain by solving complex number equation group.

Table two is the parameter of the cell to be measured by pressure channel and utilizes above-mentioned parameter to calculate acquisition Cell membrane compare electric capacity.

Table two

So far, already in connection with accompanying drawing, the present embodiment has been described in detail.According to above description, this Skilled person should characterize unicellular Young's modulus and cell membrane than electric capacity to the present invention simultaneously System has had and has clearly recognized.

Additionally, the above-mentioned definition to each element and method is not limited in the various tools mentioned in embodiment Body structure, shape or mode, it can be changed or replace by those of ordinary skill in the art simply, Such as:

(1) in addition to lock-in amplifier, impedance information acquisition module can also use electric impedance analyzer etc. Impedance measuring equipment replaces；

(2) in addition to 1kHz, low frequency can use the lower frequencies such as 2kHz (i.e. thin under this frequency The frequency that after birth condensance is corresponding much larger than cell peripheral ohmic leakage impedance) replace；Remove equally Outside 100kHz, high frequency can use the upper frequencies such as 200kHz (i.e. cell membrane capacitance under this frequency The frequency that impedance is corresponding much larger than cell peripheral ohmic leakage impedance) replace.

In sum, the present invention proposes one and characterizes unicellular Young's modulus and cell membrane compares electric capacity simultaneously System, it is achieved mechanics that cell is intrinsic and the electrology characteristic parameter i.e. Young's modulus of cell and cell membrane Gather than the high flux of electric capacity simultaneously, for cell biological physical characteristic sign provide reliable method and Approach, can be that anemia, tumor etc. exist the disease that cell biological physical characteristic changes accordingly and provide new Detection means and the new cell characteristics mark without labelling.

Particular embodiments described above, is carried out the purpose of the present invention, technical scheme and beneficial effect Further describe, be it should be understood that the foregoing is only the present invention specific embodiment and , be not limited to the present invention, all within the spirit and principles in the present invention, that is done any repaiies Change, equivalent, improvement etc., should be included within the scope of the present invention.

Claims (11)

1. one kind characterizes unicellular Young's modulus and the cell membrane system than electric capacity simultaneously, it is characterised in that including:

Micro-fluidic chip, including:

Transparent substrates,

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

Pressure channel, is formed at inside described supporting body, and 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 shooting cell to be measured under suction function by the process of described pressure channel from the lower surface of described transparent substrates；

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

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

Young's modulus obtains submodule, and the image for being shot by photographic head obtains cell to be measured and enters transient Displacements X of pressure channel_{instantaneous}With unstability displacement X_transitional, and then combine the diameter d of cell to be measured_{cell diameter}, pressure channel size W_{constriction channel}, negative pressure numerical value P_aspirationCalculate the Young's modulus of cell to be measured；

Cell membrane obtains submodule than electric capacity, and the image for being shot by photographic head obtains length L of the cell being under extended state in pressure channel_{cell elongation}, electric impedance analyzer the waveform obtained extracts the low-frequency impedance Z at pressure channel two ends when cell passes through_L0(ω) with high-frequency resistance Z_H0(ω) the low-frequency impedance Z at pressure channel two ends when, having cell to pass through_L1(ω), high-frequency resistance Z_H1, and then calculate the film of cell to be measured and compare electric capacity (ω).

System the most according to claim 1, it is characterised in that:

The material of described transparent substrates is glass or polydimethylsiloxane；

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

System the most according to claim 2, it is characterised in that described micro-fluidic chip is prepared in the following ways:

Step A, applies one layer of SU-8 5 photoresist in microscope slide Rotating with Uniform；

Step B, to the described SU-8 5 photoresist front baking on microscope slide, exposure；Do not develop, after bake, thus form pressure channel formpiston；

Step C, on described exposed SU-8 5 photoresist, Rotating with Uniform applies one layer of SU-8 25 photoresist again；

Step D, carries out front baking to described SU-8 25 photoresist, after pattern alignment figure in lithography mask version and described SU-8 5 photoresist are exposed, is exposed described SU-8 25 photoresist；

Step E, to the SU-8 25 photoresist after bake after described exposure, development；

Step F, carries out perpendicular film, forms the formpiston of the two-layer ledge structure including cell introduction passage and pressure channel described SU-8 5 and SU-8 25 photoresist；

Step G, then at performed polymer and the firming agent of described formpiston upper liquid polydimethylsiloxane；

Step H, must arrive surface have the polydimethylsiloxane block of microfluidic channel, the i.e. carrier section of chip by performed polymer and the firming agent curing and demolding of the liquid polydimethylsiloxane being cast on described formpiston；

Step I, punches described polydimethylsiloxane block so that it is through in introduction passage end positions upper and lower surface；And

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

System the most according to claim 1, it is characterised in that the cross section of described pressure channel is square, its cross-sectional area is the 40%-90% that cell cross section to be measured is long-pending.

System the most according to claim 1, it is characterised in that described negative pressure is between 100Pa to 10kPa.

System the most according to claim 1, it is characterised in that described image detection module includes:

Biological inverted microscope, its image-forming component is directed 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；

Photographic head, the biological inverted microscope of alignment is arranged, for shooting cell to be measured under suction function by the process of described pressure channel by described biological inverted microscope.

System the most according to claim 6, it is characterised in that described microscopical amplification is 400 times, the scanning speed of photographic head is 200 frames/second.

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

System the most according to claim 8, it is characterised in that described low frequency is 1kHz, described high frequency is 100kHz.

System the most according to any one of claim 1 to 9, it is characterised in that described Young's modulus obtain submodule utilize equation below calculate cell to be measured Young's modulus:

Work as d_{cell diameter}During ＜ 14.0 μm:

As 16.0 μm ＞ d_{cell diameter}During ＞ 14.0 μm:

Or, work as d_{cell diameter}During ＞ 16.0 μm:

Wherein, d_{cell diameter}For the diameter of cell to be measured, W_{constriction channel}For 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 unstability displacement, the f of pressure channel respectively_cFor the coefficient of friction of unknown pressure channel wall, W_{constriction channel}And P_aspirationFor known quantity；d_{cell diameter}、X_{instantaneous}And X_transitionalImage for being obtained by image detection module is obtained through image procossing.

11. systems according to any one of claim 1 to 9, it is characterised in that described cell membrane obtains submodule for calculating the film of cell to be measured according to following complex number equation group than electric capacity C than electric capacity_{specific membrane}:

Wherein, C_membraneFor cell membrane equivalent capacity, S_ChannelThe cross-sectional area at integration of pressure channel and introduction passage each several part is comprised for whole passage；S_{constriction Channel}Cross-sectional area for pressure channel；Z_LF0(ω)、Z_HF0(ω)、Z_LF1(ω)、Z_HF1(ω) be respectively that impedance information acquisition module collects when there is no a cell and when having cell to pass through pressure channel, the resistance value of pressure channel both sides when corresponding low frequency and high frequency；

Wherein, subscript LF represents that low frequency, HF represent high frequency, and 0 represents do not have cell, and 1 indicates cell；L_{cell elongation}Represent cell elongation length；σ_RPMIRepresent cell culture fluid electrical conductivity, C_parasiticFor system parasitic electric capacity, R_cytoplasmFor Cytoplasm equivalent resistance；

Wherein, R_mThe equivalent resistance of cell culture fluid in pressure channel when representing acellular；R_mRepresent cell by the equivalent resistance of cell culture fluid during pressure channel；R_leakRepresent the ohmic leakage formed owing to cell can not be filled up completely with pressure channel；Z_cell(ω) cell electronic circuit impedance is represented；Z_parasitic(ω) parasitic capacitance impedance is represented.