CN104122316B - Utilize distributed DC electric field to measure the mobility of particle and the method for dielectric mobility simultaneously - Google Patents

Abstract

The present invention discloses a kind of mobility that distributed DC electric field measures particle simultaneously and method of dielectric mobility utilized. The present invention makes applying direct current electric field occur distributed areas by design electrophoresis path structure, cause particle electrophoresis and dielectrophoresis coupling migration, recorded coupled motions track and the speed of particle by micro imaging system, the methods such as last Negotiation speed resolution of vectors or data fitting calculate mobility and the dielectric mobility of each particle again. In conjunction with statistical analysis, can obtain average mobility and the average dielectric mobility of similar particle thus. By measurement result of the present invention, study more accurately the surface charge property of the particles such as cell and the characteristic of molecular recognition by contributing to; With regard to cell, the movement locus of cell in the processes such as all right aid forecasting cell capture, sorting, enrichment, or optimize the particularly design of channel shape of electrophoresis system; The present invention simultaneously also provides a kind of new argument of new sign sorting particles thing.

Description

Utilize distributed DC electric field to measure the mobility of particle and the method for dielectric mobility simultaneously

Technical field

The present invention relates to a kind of mobility of particle and method of dielectric mobility simultaneously measured, be specifically related to a kind of utilization and divideCloth formula DC electric field is measured the mobility of particle and the method for dielectric mobility simultaneously.

Background technology

The mobility of the particles such as cell can be used to characterizing particles thing surface charge, is the particle surface textures such as research cellAn important physical parameter with function. In inhomogeneous field, the particles such as cell are except being subject to direct current field forceOutside effect, also there is polarization or the dielectric active force of inhomogeneous field, so occur dielectrophoresis phenomenon (Pohl, H.,Appl.Phys.1951,22,869-871.). Dielectric power size is relevant with dielectric property with particle conduction to be measured, and electrophoresis drops downDegree is that the movement velocity of mensuration particle under unit electric field intensity is similar, and dielectric mobility is to measure particle to put down in unit single gameThe dielectric speed of side under gradient (Weiss, N.G., Jones, P.V., Mahanti, P., Chen, K.P., Taylor, T.J.,Hayes, M.A.Electrophoresis2011,32,2292 – 2297.). Measuring dielectric mobility can quantitative study particleElectricity lead and dielectric property, become just gradually the particles such as cell a kind of important research new method (Weiss, N.G.,Jones,P.V.,Mahanti,P.,Chen,K.P.,Taylor,T.J.,Hayes,M.A.Electrophoresis2011,32,2292 – 2297.). But current dielectric mobility and electrophoretic mobility all separate mensuration, wherein electrophoretic mobility profitBe that uniform DC electric field is measured, its conventional measurement means comprises microscopic cells electrophoresis, Capillary Electrophoresis, swashsPhotoelectrophoresis and micro-fluid control chip electrophoretic. Dielectric mobility mainly utilizes most advanced and sophisticated principle and needle point formula electrode to induce surveyFixed as near the aggregation velocity of observation of cell under microscope electrode and the rotation of the electricity based on AC fieldElectro-rotation (ROT) technology (Watarai, H., Sakamoto, T., Tsukahara, S., Langmuir1997,13,2417-2420.). The latter's apparatus structure complexity, it is large that difficulty is controlled in operation, and non-common lab can turn round, and instituteSurvey data and apparatus parameter correlation, versatility is poor. Variety classes particle is if cell is in shape, size, surface electricalThere is extensive difference in the aspects such as lotus, cell membrane form, cell membrane character, organelle composition, polarizability, dielectric property,Derivation based on theoretical model is calculated, and process is loaded down with trivial details, is difficult to again meet the particles such as various cell and different physiology shape thereofThe needs of dielectric property research under state, only also do not have the measuring of the dielectric mobility relevant with cell itself to report at present.In addition, separately survey separately electrophoretic mobility and dielectric mobility can not disclose electric charge that particle puts at one time as cell andElectrical conductivity information, needs to develop the method that can simultaneously measure electrophoretic mobility and dielectric mobility to meet real-time dynamic studiesThe needs of the physiological status variation of cell own and cell-processes such as intermolecular quick interaction.

Summary of the invention

The object of this invention is to provide and a kind ofly utilize mobility and the dielectric that distributed DC electric field is measured particle simultaneously to drop downThe method of degree can be measured mobility and the dielectric mobility of the particles such as single or multiple cells simultaneously; The inventive method based onThe high speed microimaging electrophoresis system of distributed DC electric field, mensuration process comprises particle electrophoresis motion Position And VelocityMicro-image measure and record, and the large main process of theory analysis two of view data.

Mobility that distributed DC electric field measures particle simultaneously and the method for dielectric mobility utilized provided by the present invention,Comprise the following steps:

(1) make electrophoresis path, described electrophoresis path can form inhomogeneous DC electric field; At described electrophoresis pathEnd arranges electrolytic cell;

(2) inwall of described electrophoresis path is carried out to covalently or non-covalently chemical modification, to eliminate electric osmose;

(3) described electrolytic cell is connected with dc source, forms electrophoresis system; Described electrophoresis system is placed in micro-On video recording observation platform, and the partial adjustment that described electrophoresis path is produced to inhomogeneous DC electric field is in microscopic field;

(4) in a described electrolytic cell, add particle suspension liquid, and make grain flow to microscopic field; To all the other instituteState and in electrolytic cell, add background electrolyte, and flow in described electrophoresis path; Control the liquid level in described electrolytic cellHighly equal;

(5) between the described electrolytic cell of placing the described electrolytic cell of described suspended particulate substance and be oppositely arranged, apply direct currentVoltage; Utilize video record system to observe and record movement locus and the speed of particle under particular field strength;

(6) method of utilizing finite element electric field to simulate, the DC electric field calculating in micro-focal plane distributes, respectively surveysThe electric-field intensity of fixed point and the gradient of electric field strength square;

(7) utilize following 1) or 2) mobility and the dielectric mobility of method count particles;

1) utilize formula (1) to obtain the mobility of particle;

μ_EP＝υ_EP/ E; Formula (1)

In formula (1), μ_EPRepresent mobility, υ_EPRepresent electrophoretic velocity; E represents shimming field intensity;

υ_EPThe moving track calculation of the particle being obtained by step (5) obtains;

Utilize formula (2) to obtain the dielectric mobility of particle;

$\mathrm{\υ}={\mathrm{\μ}}_{\mathrm{EP}}E+{\mathrm{\μ}}_{\mathrm{DEP}}\▿\left(E^2\right);$ Formula (2)

In formula (2), μ_EPRepresent mobility, E represents shimming field intensity, μ_DEPRepresent dielectric mobility,Represent unevenThe gradient of even electric field strength square, υ represents the movement velocity of particle;

The moving track calculation of the particle that υ is obtained by step (5) obtains;

2) utilize formula (2) to carry out data fitting, obtain mobility and the dielectric mobility of particle;

$\mathrm{\υ}={\mathrm{\μ}}_{\mathrm{EP}}E+{\mathrm{\μ}}_{\mathrm{DEP}}\▿\left(E^2\right);$ Formula (2)

In formula (2), μ_EPRepresent mobility, E represents shimming field intensity, μ_DEPRepresent dielectric mobility,Represent unevenThe gradient of even electric field strength square, υ represents the movement velocity of particle;

The process of described data fitting is as follows:

The movement locus that utilizes the particle that step (5) obtains, calculates on the power line of at least 2 non-uniform electric fieldsThe movement velocity of particle, obtains the equation group about mobility and dielectric mobility;

Obtain the equation group of at least 2 groups about mobility and dielectric mobility according to above-mentioned steps;

By the data fitting to each equation group, obtain mobility and the dielectric mobility of particle.

In above-mentioned method, described electrophoresis path is realized the object that forms inhomogeneous DC electric field by following manner:

1) expansion of caliber or dwindle;

2) bending of stream;

3) intersection of stream.

In above-mentioned method, described electrophoresis path specifically can be following any:

1) cruciform shape passage;

2) straight shape passage is provided with symmetrical hemisphere jut.

In above-mentioned method, if the biologic grain thing such as particle to be measured is cell, described background electrolyte should be chosen asDeng the physiological buffer electrolyte solution oozing.

In above-mentioned method, the material of making described electrophoresis path can be glass, quartz, lucite, MerlonOr dimethyl silicone polymer.

In above-mentioned method, utilize glass to make described electrophoresis path, described electrophoresis path is carried out to following chemistry and repairDecorations:

1) clean described electrophoresis path with sodium hydrate aqueous solution, water, hydrochloric acid and water successively;

2) in described electrophoresis path, pour into γ-(methacryloxypropyl) oxypropyl trimethyl oxosilane colloidal sol; Leave standstill 30minAfter, water cleans described electrophoresis path;

3) in described electrophoresis path, pour into pre-gathering solutions, described pre-gathering solutions by acrylamide, ammonium persulfate,N, N, N', N'-tetramethylethylenediamine and water composition, wherein, the quality percentage composition of described acrylamide is 2%, described inThe quality percentage composition of ammonium persulfate is 10%, described N, and N, N', the quality percentage composition of N'-tetramethylethylenediamine is10%; After leaving standstill placement, water cleans described electrophoresis path.

The inventive method can be used for measuring mobility and the dielectric mobility of the biologic grain such as cell or bacterium simultaneously.

In the inventive method, the video record system of stating adopting can be high-speed CCD.

Utilize the inventive method to obtain mobility and the dielectric mobility data of individual particle, then get final product by statistical analysisTo average mobility and the average dielectric mobility of similar particle.

The present invention utilizes distributed DC electric field, has realized single or multiple particles and has dropped down as the electrophoretic mobility of cell and dielectricWhen degree, measure. By measurement result of the present invention, the surface of the particles such as cell will be contributed to study more accuratelyThe characteristic of charge property and molecular recognition; With regard to cell, all right aid forecasting cell capture, sorting, enrichment etc.The movement locus of cell in process, or optimize the particularly design of channel shape of electrophoresis system; The present invention simultaneously also providesA kind of new argument of new sign sorting particles thing.

Brief description of the drawings

Fig. 1 measures the experimental provision schematic diagram of mobility and the dielectric mobility of cell simultaneously with distributed DC electric field;

Fig. 2 is for building " ten " word chi structure schematic diagram of distributed DC electric field;

Fig. 3 is for building the blistered structural representation of distributed DC electric field;

Fig. 4 is finite element modelling " ten " word chi structure distributed DC electric field intensity and electric field line distribution figure;

Fig. 5 is the blistered structure distribution formula of finite element modelling DC electric field electric-field intensity and electric field line distribution figure;

Fig. 6 is single human red cell electrophoresis sequence fortune by " ten " word cross aisle distributed DC electric field CCD is taken the photographMotion video;

Fig. 7 is that in " ten " word cross aisle distributed DC electric field, single human red cell dielectric speed becomes with x coordinateChange figure;

Fig. 8 is " ten " least square method data fitting income earner erythrocyte mobility (Fig. 8 (a)) and dielectric mobility(Fig. 8 (b)) distribution map.

Detailed description of the invention

The experimental technique using in following embodiment if no special instructions, is conventional method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

In following embodiment, microscopical model used is OLYMPUSIX71 inverted fluorescence microscope.

Embodiment 1, utilization " ten " word cross aisle are measured cell mobility and dielectric mobility simultaneously

(1) design and fabrication of " ten " word cross channel structure device

Select glass material to build right-angled intersection passage, channel size as shown in Figure 2, the construction method of passage and photoetchingLegal system is identical for the preparation method of glass micro-fluidic chips, comprises mask design, making, uv-exposure (15s), hydrogenFluoric acid etching (25 DEG C, HF – HNO₃–H₂Etching 20min in O solution), the step such as involution, punching.

(2) chemical modification of vias inner walls

Use successively 1MNaOH, water, 1MHCl and water flushing channel, then pour into γ-(methacryloxypropyl) thirdBase trimethyl oxosilane colloidal sol, after 30min, water flushing channel, then pour into freshly prepd pre-gathering solutions, pre-polymerization is moltenLiquid is by the N of the ammonium persulfate and 10% (w/w) of the acrylamide, 10% (w/w) of 2% (w/w), N, N', N'-Tetramethylethylenediamine composition, room temperature is placed after 4h, and water flushing channel is dried.

(3) electrophoresis experiment

" ten " word cross channel structure device is connected with power supply, forms electrophoresis system, and be placed in micro-video recording observationOn platform, " ten " word crossing is adjusted in microscopic field; PBS cushioning liquid (pH7.4) is done in capillaryWith under be circulated in passage, leave standstill after balance, get the healthy Human red blood cells in suspensions displacement of 5 μ L buffer solutions, then toIn other liquid storage tank, respectively add the background electrolyte (1.9mMNa of 5 μ L₂HPO₄，8.1mMKH₂PO₄，0.85％NaCl), and the liquid level of adjusting each liquid storage tank at field range inner cell in static, apply 50V direct currentPress. In whole experimentation, should check that passage and each slot electrode are to guarantee not having Bubble formation.

Fig. 1 is the above-mentioned experimental provision schematic diagram of constructing.

(4) finite element electric field simulation

The numerical simulation of the Electric Field Distribution in two dimension cell focussing plane is to obtain based on Finite Element Method.

Model forms the needs for adapting to physical channel by two rectangles. Simulation obtains the electric-field strength of each NodesAfter degree, can obtain the electric-field intensity of the arbitrary position of passage by electric-field intensity is carried out to interpolation, and then obtain arbitrarilyThe gradient of the electric-field intensity of any square.

Fig. 4 is electric-field intensity and electric field line distribution figure in finite element modelling " ten " word cross aisle.

(5) tracking cell and data processing

Fig. 6 is taken the photograph single human red cell electrophoresis sequence motion by " ten " word cross aisle distributed DC electric field CCDImage.

The track of cell is to take the photograph human red cell electrophoresis order by self-editing shell script from distributed DC electric field CCDIn row moving image, extract cell position information.

Ask first derivative to obtain cell movement along the total electromigration speed v of x direction of principal axis to the time cell position. For terribleTo mobility and the dielectric mobility of cell in individual cells track, calculate by following two kinds of modes:

First kind of way is to utilize formula (1) to obtain the cell mobility of uniform electric field part,

μ_EP＝υ_EP/ E; Formula (1)

In formula (1), μ_EPRepresent mobility, υ_EPRepresent electrophoretic velocity; E represents shimming field intensity;

In non-equal electric field, the poor cell electrophoresis speed that deducts of cell coron velocity obtains cell dielectric speed, and Fig. 7 isSingle human red cell dielectric speed is with x changes in coordinates figure. Dielectric speed is divided by cell movement track place electric field square ladderDegree obtains cell dielectric mobility.

The second way is by with following formula (2):

$\mathrm{\υ}={\mathrm{\υ}}_{\mathrm{EP}}+{\mathrm{\υ}}_{\mathrm{DEP}}={\mathrm{\μ}}_{\mathrm{EP}}E+{\mathrm{\μ}}_{\mathrm{DEP}}\▿\left(E^2\right)$

Cell movement general speed on each track is carried out to least square fitting, by μ_EPAnd μ_DEP(representDielectric mobility) be optimized, so that must calculate cell movement general speed and test the cell movement general speed obtainingDeviation minimum between value, thus cell mobility and the dielectric mobility on individual cells track obtained. Then pass through instituteHave the mobility data on track to add up, the mobility and the dielectric mobility that obtain cell distribute and mean value, institute's scoreCloth the results are shown in Figure 8.

Embodiment 2, utilize spherical bubbling capillary to measure cell mobility and dielectric mobility simultaneously

(1) passage is made:

In 100 microns of internal diameter spring glass capillaries, heating and melting blows that to be drawn into radius size be the ball bubble of 120 micronsShape structure, access diagram as shown in Figure 3.

(2) passage method of modifying is with embodiment 1.

(3) electrophoresis experiment

Be on PDMS pad, to make 1cm × 1cm reservoir at dimethyl silicone polymer, capillary port is insertedIn PDMS pad to be connected with Buffer Pool. PBS cushioning liquid (pH7.4) is joined in reservoir to solutionUnder capillarity, be circulated in capillary, leave standstill after balance, get the healthy Human red blood cells in suspension displacement of 5 μ L bufferingLiquid then adds the background electrolyte of 5 μ L in other end capillary connects reservoir, and adjusts each liquid storage tankLiquid level at field range inner cell in static; Apply 50V DC voltage. In whole experimentation, should examineLook into capillary and two end electrodes groove and guarantee there is no Bubble formation.

(4) finite element (FiniteElementAnalysis, FEA) numerical Simulation of Electric Field

The numerical simulation of the Electric Field Distribution in two dimension cell focussing plane is to obtain based on Finite Element Method equally. Model isForm the needs for adapting to physical channel by two rectangles and spherical structure. The 10mMPBS cushioning liquid of pH7.4Electrical conductivity is 13.00mS/cm. The voltage of passage left end is made as to 0, and passage right-hand member is made as by experimental voltage geometric ratio and calculatesMagnitude of voltage. Simulation obtains after the electric-field intensity of each Nodes, can obtain by electric-field intensity is carried out to two-dimensional interpolationTo the electric-field intensity of the arbitrary position of passage, and then obtain the gradient of the electric-field intensity square of any point.

Fig. 5 is electric-field intensity and electric field line distribution figure in capillary ball balloon-shaped structure.

Data handling procedure and method are with embodiment 1.

Claims (7)

1. utilize distributed DC electric field to measure the mobility of particle and a method for dielectric mobility simultaneously, comprise followingStep:

(1) make electrophoresis path, described electrophoresis path can form inhomogeneous DC electric field; At described electrophoresis pathEnd arranges electrolytic cell;

(2) inwall of described electrophoresis path is carried out to covalently or non-covalently chemical modification, to eliminate electric osmose;

(3) described electrolytic cell is connected with dc source, forms electrophoresis system; Described electrophoresis system is placed in micro-On video recording observation platform, and the partial adjustment that described electrophoresis path is produced to inhomogeneous DC electric field is in microscopic field;

(4) in a described electrolytic cell, add particle suspension liquid, and make grain flow to microscopic field; To all the other instituteState and in electrolytic cell, add background electrolyte, and flow in described electrophoresis path; Control the liquid level in described electrolytic cellHighly equal;

(5) between the described electrolytic cell of placing the described electrolytic cell of described suspended particulate substance and be oppositely arranged, apply direct currentVoltage; Utilize video record system to observe and record movement locus and the speed of particle under particular field strength;

(6) method of utilizing finite element electric field to simulate, the DC electric field calculating in micro-focal plane distributes, respectively surveysThe electric-field intensity of fixed point and the gradient of electric field strength square;

(7) utilize following 1) or 2) mobility and the dielectric mobility of method count particles;

1) utilize formula (1) to obtain the mobility of particle;

μ_EP＝υ_EP/ E; Formula (1)

In formula (1), μ_EPRepresent mobility, υ_EPRepresent electrophoretic velocity; E represents shimming field intensity;

υ_EPThe moving track calculation of the particle being obtained by step (5) obtains;

Utilize formula (2) to obtain the dielectric mobility of particle;

$\mathrm{\υ}={\mathrm{\μ}}_{EP}E+{\mathrm{\μ}}_{DEP}\▿\left(E^2\right);$ Formula (2)

In formula (2), μ_EPRepresent mobility, E represents shimming field intensity, μ_DEPRepresent dielectric mobility,Represent unevenThe gradient of even electric field strength square, υ represents the movement velocity of particle;

The moving track calculation of the particle that υ is obtained by step (5) obtains;

2) utilize formula (2) to carry out data fitting, obtain mobility and the dielectric mobility of particle;

$\mathrm{\υ}={\mathrm{\μ}}_{EP}E+{\mathrm{\μ}}_{DEP}\▿\left(E^2\right);$ Formula (2)

In formula (2), μ_EPRepresent mobility, E represents shimming field intensity, μ_DEPRepresent dielectric mobility,Represent unevenThe gradient of even electric field strength square, υ represents the movement velocity of particle;

The process of described data fitting is as follows:

The movement locus that utilizes the particle that step (5) obtains, calculates on the power line of at least 2 non-uniform electric fieldsThe movement velocity of particle, obtains the equation group about mobility and dielectric mobility;

Obtain the equation group of at least 2 groups about mobility and dielectric mobility according to above-mentioned steps;

By the data fitting to each equation group, obtain mobility and the dielectric mobility of particle.

2. method according to claim 1, is characterized in that: described electrophoresis path is realized shape by following mannerBecome the object of inhomogeneous DC electric field:

1) expansion of caliber or dwindle;

2) bending of stream;

3) intersection of stream.

3. method according to claim 2, is characterized in that: described electrophoresis path be following any:

1) cruciform shape passage;

2) straight shape passage is provided with symmetrical hemisphere jut.

4. according to the method described in any one in claim 1-3, it is characterized in that: the material of making described electrophoresis pathMatter is glass, quartz, lucite, Merlon or dimethyl silicone polymer.

5. method according to claim 4, is characterized in that: utilize glass to make described electrophoresis path, to instituteState electrophoresis path and carry out following chemical modification:

1) clean described electrophoresis path with sodium hydrate aqueous solution, water, hydrochloric acid and water successively;

2) in described electrophoresis path, pour into γ-(methacryloxypropyl) oxypropyl trimethyl oxosilane colloidal sol; Leave standstill 30minAfter, water cleans described electrophoresis path;

3) in described electrophoresis path, pour into pre-gathering solutions, described pre-gathering solutions by acrylamide, ammonium persulfate,N, N, N', N'-tetramethylethylenediamine and water composition, wherein, the quality percentage composition of described acrylamide is 2%, described inThe quality percentage composition of ammonium persulfate is 10%, described N, and N, N', the quality percentage composition of N'-tetramethylethylenediamine is10%; After leaving standstill placement, water cleans described electrophoresis path.

6. method according to claim 5, is characterized in that: described particle is cell or bacterium.

7. method according to claim 6, is characterized in that: in step (4), and described video record systemFor CCD.