CN101275944B - High flux test chip of single cell traveling wave dielectric spectrum and test method - Google Patents
High flux test chip of single cell traveling wave dielectric spectrum and test method Download PDFInfo
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Abstract
A method for testing a high-pass test chip of the single-cell travelling wave dielectric spectrum comprises the following procedures: respectively connecting each terminal of the test chip to the source end of the sinusoidal voltage signal which has same frequency to lead to that the advance mount or hysteresis amount of the phase between random two adjacent electrodes is equal, dripping the liquid sample containing the cell to be tested into the inner part of the test chip from the injection port (06), then projecting light pattern to the cell initial collection area (32) at the central area of the electrode group (10), projecting a plurality of strip light patterns (31) with width about the cell diameter from the lower part of the chip substrate (01) to the microelectrode group (10) to form a virtual electrode array, regulating the frequency of a waveform generator, and at the same time recording the horizontal moving image of all cells in the frequency band through a CCD and an image recorder on a microscope. If the cell number of the completed test actually has already satisfied the requirement of the test, then the test procedure ends.
Description
Technical field
The present invention relates to the concurrent testing technology of unicellular dielectric response and the technology of controlling of micro-and nano-particles.
Background technology
According to Maxwell-Wagner interfacial polarization theory as can be known, cell add will be passive under the AC field condition generation polarization, cause the dielectric relaxation phenomenon of cell.The DIELECTRIC CONSTANT ε of cell and conductivityσ can draw according to feature and cell frequency domain passive characteristic (or claiming dielectric spectra) parsing of dielectric relaxation.Research to cell frequency domain passive characteristic has become one of study hotspot content of cell electrophysiology and cell biophysics.Measure the interior dielectric response of frequency domain of cell and compose the sign that can realize cell.
The method of obtaining at present cell dielectric characteristic or dielectric spectra has internal electrode method, gap method, AC impedence method, electric wheel measuring method, Micropipet method etc., wherein electricity rotation dielectrophoresis mensuration can obtain the frequency characteristic of imaginary part of the Clausius-Mossotti factor (the CM factor) of cell, the method is as a kind of non-destructive technology, simple with its electrode structure, be convenient to miniaturization and integrated, be easy to realize that the advantage such as automatically control obtains widespread use and also realized commercialization.But present electric rotary test chip and method of testing thereof are unsatisfactory, at first, the electricity wheel measuring be the angular velocity of rotation of cell, for the optical appearance uniformity or have for the cell of circumference symmetrical feature, the difficulty of using machine vision technique automatically to detect strengthens; Secondly, in order to test simultaneously a plurality of samples, usually have a plurality of electric rotation electrode groups on the electrorotation chip, form the electrode group pattern, and need between the electrode group to keep certain spacing in order to avoid mix between the sample, it is loose that this just causes cell to be measured to arrange, so the sample size that can test in the limited test space is limited, is unfavorable for realizing high-throughout test; The 3rd, the roll rate of the cell of diverse location does not have direct comparability in the electric rotating drum, and is subject to the interference of bottom surface friction force, and measuring accuracy is low, and poor stability is not suitable for the test of single celled high-flux parallel, the sample introduction of cell and separate yet relatively more difficult.Although the electricity wheel measuring can fix unicellular its System of Rotating about Fixed Axis that makes by means of the light tweezer, but the light tweezer need to converge by force laser beam and be not suitable for catching for a long time of cell, be easy to cause cellular damage, and laser beam can cause cell peripheral fluid local heating, form convection current, and then affect the electric wheel measuring precision of cell; In addition, light tweezer moving range is very little, is not suitable for single celled large-scale parallel and catches and control, and can't realize simultaneously cell sample introduction, test.And the introduction of light tweezer is not easy to promote the use of so that the cost of whole test macro sharply rises.
Row ripple dielectrophoresis is as the variant of electricity rotation dielectrophoresis, be equivalent to the electric rotation electrode generate straight line with circle distribution, so can obtain equally the imaginary part of the CM factor of cell by the point-to-point speed that detects cell, and the detection of point-to-point speed is easy more than spin angle velocity, reliability and stability will promote, degree of dependence to the optical appearance of cell reduces greatly, and applicable cell category will be more extensive; In addition, it is very tight that the traveling wave electrode array can be arranged, and forms stronger electric field intensity, will make the dielectric characterization of cell (the especially small especially cell of volume) more obvious.From above-mentioned angle, the potentiality that row ripple dielectrophoresis is used for the cell dielectric spectroscopy test are very large.Yet, although row ripple dielectrophoresis has above-mentioned advantage, the normally motion of cell colony of response effect of row ripple dielectrophoresis, the position relationship between cell and the cell be at random and uncontrollable, be difficult to still realize that the high-flux parallel of unicellular dielectric spectrum tests.Come subtest even introduce the light tweezer, still have the problem same with above-mentioned electric rotary test.
Therefore, on the basis of the advantage of inheriting above line ripple dielectrophoresis, how in a big way, to control flexibly the position relationship between each cell, and then dielectric response is the key issue that realizes the high-flux parallel test of unicellular dielectric spectrum to make each cell produce separately independently.
Given this, the present invention proposes a kind of high flux test chip and method of testing of single cell traveling wave dielectric spectrum, be convenient to Machine Vision Detection in the response of succession row ripple dielectrophoresis, and on the advantage basis of electrode group structure compactness, be integrated on the chip that monobasal drives with traditional capable ripple microelectrode with based on the optical mode dummy electrodes of photoconductive principle, by to the suitable light pattern of chip electrode group planar projective, position relationship on a large scale between each cell of control, and make each cell produce separately independently dielectric response, to solve electric rotary test technology to the automatic detection difficult of cell with the symmetrical optical appearance of circumference, each test sample book quantity is few, and light tweezer moving range is little can not realize the problem of concurrent testing on a large scale.
Summary of the invention
Technical matters: the high flux test chip and the method for testing that the purpose of this invention is to provide single cell traveling wave dielectric spectrum, can make separately dielectric response independently of each unicellular generation to be measured, and then realize high-throughout concurrent testing, to solve present cell dielectric response measuring technology to the automatic detection difficult of cell with the symmetrical optical appearance of circumference, each test sample book quantity is few, and light tweezer moving range is little and can not realize the problem of concurrent testing on a large scale.
Technical scheme: the high flux test chip and the method for testing that the invention provides a kind of single cell traveling wave dielectric spectrum, use the microelectrode of three-decker to consist of the traveling wave electrode group, physical electrode and optical mode dummy electrodes are integrated in one single chip, realizing the complementarity of the two, and then finish the measurement of single celled row ripple dielectric response spectrum.
For reaching above-mentioned technical purpose, the technical solution used in the present invention is:
The high flux test chip of single cell traveling wave dielectric spectrum provided by the invention comprises microelectrode group, chip base, conductive layer, the insulation course with via hole, chip middle ware interlayer, chip cover plate and injection port; Be provided with conductive layer in chip base, be provided with insulation course with via hole at conductive layer, be provided with wall at the edge with the insulation course of via hole, the insulation course with via hole in chip middle ware interlayer is provided with the microelectrode group, be provided with the chip cover plate at chip middle ware interlayer, injection port is located at the middle part of chip cover plate.The material of described chip base, insulation course, chip middle ware interlayer and chip cover plate is insulating material.Described insulating material comprises glass or silicon or high molecular polymer or their insulating material that is combined in interior good biocompatibility.The microelectrode group is comprised of many parallel elongate strip electrodes, and the formation cycle traveling wave electrode of arranging, and all electrodes that produce same potential are connected to same terminal, and electrode directly is connected to conductive layer or the via hole by insulation course is connected to conductive layer.When conductive layer was transparent conductive material, conductive layer covered whole test section.Each microelectrode in the microelectrode group is divided into three layers on intrinsic photo-conductivity layer, n+ type photoconductive layer, transparent conductive film layer; The material of photoconductive layer comprises the material with photoconductive property of amorphous silicon hydride; The material of transparent conductive film layer comprises the conductive material with high transmission rate of tin indium oxide.Chip base and chip cover plate must be good transparent insulation materials, are beneficial to the transmission of light pattern and the observation of cell movement.
The high flux method of testing of single cell traveling wave dielectric spectrum provided by the invention is:
Step 1: each terminal of test chip is connected respectively to the sine voltage signal source with same frequency, so that the phase place between any two adjacent electrodes is leading or hysteresis all equates,
Step 2: the liquid sample that will contain cell to be measured splashes into test chip inside from injection port, then the cell in electrode group switching centre zone initially gathers district's projection light pattern, this zone is illuminated fully, signal to test chip input small magnitude, pass through regulating frequency, make the cell mass at injection port place produce among a small circle motion, and then cell is spread out
Step 3: direction microelectrode group is throwed the strip light pattern that a plurality of width are about cell dia under the chip base, form the dummy electrodes array, and make it lay respectively at the both sides in cell initial aggregation district, adjust the distance between each strip light pattern and the cell initial aggregation district, so that each strip light pattern contains a cell;
Step 4: the frequency of regulating waveform generator, simultaneously by the CCD on the microscope and image recorder with under the tangential movement recording image of all cells in above-mentioned frequency range, process to draw the movement velocity of each cell with the change curve of exciting signal frequency through image;
Step 5: if the actual cell quantity of having finished test can satisfy testing requirement, then test process finishes; Otherwise, must be with each strip light patterns of the both sides, cell initial aggregation district described in the step 3 to the center translation, so that remaining cell to be measured is in the elongate strip light pattern, then repeating step 4, until satisfy the quantity demand of test cell.
The virtual electric array that described strip light pattern forms on chip electrode group plane comprises equidistant array, not equidistant array or the dummy electrodes array of both combinations.
The capable ripple microelectrode group of using photoconductive material to consist of produces row ripple electric field, the light pattern that uses simultaneously dummy electrodes direct-writing device (obtain picture signal and generate the device of micro pattern from computing machine) to be scheduled in the projection of chip electrode group plane.According to photoconductive effect, the photo-conductive film material has high resistance value in the situation of irradiation not, and its electric conductivity strengthens greatly when accepting external light source, therefore, when light image projects on the photoconductive layer, originally the medium of approximate insulation almost becomes conductor, namely formed the dummy electrodes array, so that the relative position relation between each cell is controlled, and then so that each cell advance according to optical mode path level separately respectively, to realize the high-flux parallel test, then by changing the frequency of signal generation apparatus, record the motion conditions of all cells to be measured of each Frequency point by image collecting device, use at last image processing method to draw the movement velocity of each cell, and then obtain the dielectric spectra of each cell in the certain frequency scope.In test process, the shape of dummy electrodes array is that the light pattern by projection determines therefore to have very high flexibility, and can Real-time Reconstruction, can adjust flexibly as the case may be shape, size and the position of dummy electrodes array.
Beneficial effect: the present invention proposes high flux test chip and the method for testing of single cell traveling wave dielectric spectrum, respond on the basis of the advantage of being convenient to Machine Vision Detection and electrode group structure compactness thereof at succession row ripple dielectrophoresis, optical mode dummy electrodes and traditional physics microelectrode are integrated on the chip of monobasal driving, can realize the high-flux parallel test request of unicellular dielectric spectrum.Compare with the electric rotation test method that electric rotary test chip and light tweezer are assisted, the present invention realizes the concurrent testing of a plurality of cells by making up optical mode dummy electrodes array, overcome the little defective that can not realize that large-scale parallel is controlled of light tweezer moving range; The foundation of method of testing provided by the invention is photoconductive principle, the energy source of excitation cell movement is not from radiation source in signal generation apparatus, and this and light tweezer have essential distinction, therefore can be because of illumination damaging cells, overcome the light tweezer and caught for a long time damage problem to living cells; The substrate of test chip provided by the invention is sandwich construction, is convenient to the connection of each microelectrode, and chip pin is few, and is easy to connect with external device (ED); Test chip provided by the invention is all applicable to the situation that positive and negative dielectrophoresis occurs cell, and can finish the pretreatment process such as fine position before sample introduction, the test.In addition, test chip provided by the invention is that monobasal drives, and needs the device of double-basis plate power supply than some, and is easier to be integrated with other microfluidic device.
Description of drawings
Fig. 1 is the structural representation (only drawing a few microelectrode and amplification to illustrate) of the high flux test chip of single cell traveling wave dielectric spectrum provided by the invention;
Fig. 2 is the vertical view (electrode group structure has the cycle characteristic of arranging, and only draws several cycles here to illustrate) of the high flux test chip of single cell traveling wave dielectric spectrum provided by the invention;
Fig. 3 is the synoptic diagram after the high flux test chip of single cell traveling wave dielectric spectrum provided by the invention loads optical mode dummy electrodes array, and the zone that the dotted line frame among the figure surrounds is the light area.
Embodiment
The embodiment of the high flux test chip of single cell traveling wave dielectric spectrum provided by the invention comprises microelectrode group 10, chip base 01, conductive layer 02, the insulation course 03 with via hole, chip middle ware interlayer 04, chip cover plate 05 and injection port 06 as depicted in figs. 1 and 2.Microelectrode group 10 is divided into three layers: intrinsic photo-conductivity layer 11, n+ type photoconductive layer 12 and transparent conductive film layer 13.The material of intrinsic photo-conductivity layer 11 can be selected the eigenstate amorphous silicon hydride; The material of n+ type photoconductive layer 12 can be selected n+ type amorphous silicon hydride; The material of transparent conductive film layer 13 can be selected the indium tin oxide transparent membraneous material; The width of electrode generally should be more than 10 times of thickness of electrode, and electrode spread should be tried one's best closely.The material of chip base 01, chip middle ware interlayer 04 can be dimethyl silicone polymer or SU-8 photoresist.Conductive layer 02 forms the first electrode leading-out terminal 021, the second electrode leading-out terminal 022, third electrode leading-out terminal 023, the 4th electrode leading-out terminal 024 in chip periphery; On the insulation course 03 with via hole, its objective is those are not connected to following conductive layer 02 with the electrode that the 4th electrode leading-out terminal 024 is connected by via hole with the second electrode leading-out terminal 022, and then is connected with the third electrode leading-out terminal with the first electrode leading-out terminal 021 and is connected; For insulation course 03 be positioned at conductive layer 02 below it, if opaque material then should be tried one's best into long narrow bar shaped (referring to Fig. 2) in order to avoid cover light impact observation, but if the good material of the transparency, just can cover larger zone, or even whole test section.In the present embodiment, chip has 3 phase input signals, has the input signal of two terminals identical in the electrode leading-out terminal (i.e. in parallel the use), mainly is the pressure drop that causes in order to reduce elongated conductive layer.Injection port 06 is positioned at the middle section of chip cover plate, so that different cells is moved in the opposite direction by the central area of chip.
The embodiment of the high flux method of testing of single cell traveling wave dielectric spectrum provided by the invention is referring to Fig. 3, use photoconductive material to form the elongated strip shaped microelectrode array and produce row ripple electric field, use simultaneously the dummy electrodes direct-writing device at the predetermined light pattern array of chip electrode group plane projection, and then control relative position relation between each cell, and so that each cell advance according to optical mode path level separately respectively, to realize the high-flux parallel test, then by changing signal frequency, record the motion conditions of all cells to be measured of each Frequency point by image collecting device, use at last image processing method to draw the point-to-point speed of cell, and then obtain the dielectric spectra of each cell in the certain frequency scope.Wherein, the dummy electrodes direct-writing device obtains picture signal from computing machine and generates light pattern through Digital Micromirror Device (DMD) drive plate and form small light pattern and then project tiny area on the chip through the lens optical path unit; Image collecting device comprises microscope, ccd video camera and image acquisition converting unit, and its output terminal is connected with computing machine.
The concrete steps of the embodiment of the high flux method of testing of single cell traveling wave dielectric spectrum provided by the invention are as follows:
Step 1: four terminals of chip are connected respectively to the sine voltage signal source with same frequency.Wherein terminal 022 input initial phase is the signal of 0 degree; Terminal 021 and 023 input initial phase are the signal of 120 degree, and the purpose that these two terminals connect same signal end is the pressure drop effect that reduces the long distance of elongated conductive layer; Terminal 024 input initial phase is the signal of 240 degree.Regions of electrode groups on the chip can produce capable ripple electric field, can advance perpendicular to the direction of electrode on the inducing cell edge;
Step 2: the liquid sample that will contain cell to be measured with injector splashes into chip internal from injection port 06, and at this moment, a large amount of cells are collected in the among a small circle zone of electrode group switching centre, i.e. cell initial aggregation district 32 (referring to Fig. 3).At cell initial aggregation district 32 projection light patterns, this zone is illuminated fully, and opens the signal of waveform generator output small magnitude, by regulating frequency, make cell mass produce among a small circle motion, and try to regulate cell is spread out.
Step 3: direction microelectrode group projection width is about a plurality of elongated strip shaped light patterns 31 (referring to Fig. 3) of cell dia under the chip base, and light pattern 31 lays respectively at the both sides in cell initial aggregation district 32.The cell that only is in inside, light area is just understood the occurred level motion, and unicellular meeting is advanced along the path that the elongated strip shaped area of illumination forms; The cell quantity of measuring according to need during test is set the quantity of elongate strip light pattern, forms a plurality of parallel elongate bar light patterns, to realize the parallel measurement of a plurality of cells.
Step 4: the frequency of regulating waveform generator to the scope of hundreds of MHz at tens KHz, simultaneously by the CCD on the microscope and image recorder with all cells in above-mentioned frequency range under the tangential movement recording image, draw the movement velocity of each cell with the change curve of exciting signal frequency through image processing and speed detection algorithm, per 10 octaves are got 4～8 test points usually;
Step 5: if the actual cell quantity of having finished test can satisfy testing requirement, then test process finishes; Otherwise, must be with the elongate strip light pattern array of 32 both sides, cell initial aggregation district described in the step 3 to the center translation, so that remaining cell to be measured is in the elongate strip light pattern, then repeating step 4, until satisfy the quantity demand of test cell.
Claims (8)
1. the high flux test chip of a single cell traveling wave dielectric spectrum, it is characterized in that this test chip comprises microelectrode group (10), chip base (01), conductive layer (02), the insulation course (03) with via hole, chip middle ware interlayer (04), chip cover plate (05) and injection port (06); Be provided with conductive layer (02) in chip base (01), be provided with insulation course (03) with via hole at conductive layer (02), be provided with chip middle ware interlayer (04) at the edge with the insulation course (03) of via hole, the insulation course with via hole in chip middle ware interlayer (04) (03) is provided with microelectrode group (10), be provided with chip cover plate (05) at chip middle ware interlayer (04), injection port (06) is located at the middle part of chip cover plate (05).
2. the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 1, it is characterized in that the material of described chip base (01), insulation course (03), chip middle ware interlayer (04) and chip cover plate (05) is insulating material.
3. the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 2 is characterized in that, described insulating material comprises glass or silicon or high molecular polymer or their insulating material that is combined in interior good biocompatibility.
4. the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 1, it is characterized in that, microelectrode group (10) is comprised of many parallel elongate strip electrodes, and the formation cycle traveling wave electrode of arranging, and all electrodes that produce same potential are connected to same terminal, and electrode directly is connected to conductive layer (02) or the via hole by insulation course (03) is connected to conductive layer (02).
5. the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 1 is characterized in that, when conductive layer (02) was transparent conductive material, conductive layer (02) covered whole test section.
6. the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 1, it is characterized in that each microelectrode in the microelectrode group (10) is divided into (13) three layers on intrinsic photo-conductivity layer (11), n+ type photoconductive layer (12), transparent conductive film layer; The material of intrinsic photo-conductivity layer (11) comprises the material with photoconductive property of amorphous silicon hydride; The material of transparent conductive film layer (13) comprises the conductive material with high transmission rate of tin indium oxide.
7. the method for testing of the high flux test chip of a single cell traveling wave dielectric spectrum as claimed in claim 1 is characterized in that this method of testing is:
Step 1: each terminal of test chip is connected respectively to the sine voltage signal source with same frequency, so that the phase place between any two adjacent electrodes is leading or hysteresis all equates,
Step 2: the liquid sample that will contain cell to be measured splashes into test chip inside from injection port (06), then the cell in electrode group (10) central area initially gathers district (32) projection light pattern, this zone is illuminated fully, signal to test chip input small magnitude, pass through regulating frequency, make the cell mass at injection port place produce among a small circle motion, and then cell is spread out
Step 3: the strip light pattern (31) that is about cell dia from a plurality of width of lower direction microelectrode group (10) projection of chip base (01), form the dummy electrodes array, and make it lay respectively at the both sides in cell initial aggregation district (32), adjust the distance between each strip light pattern (31) and the cell initial aggregation district (32), so that each strip light pattern contains a cell;
Step 4: the frequency of regulating waveform generator, simultaneously by the CCD on the microscope and image recorder with under the tangential movement recording image of all cells in above-mentioned frequency range, process to draw the movement velocity of each cell with the change curve of exciting signal frequency through image;
Step 5: if the actual cell quantity of having finished test can satisfy testing requirement, then test process finishes; Otherwise, must be with each strip light patterns (31) of the both sides, cell initial aggregation district (32) described in the step 3 to the center translation, so that remaining cell to be measured is in the elongate strip light pattern, then repeating step 4, until satisfy the quantity demand of test cell.
8. the method for testing of the high flux test chip of single cell traveling wave dielectric spectrum as claimed in claim 7 is characterized in that the virtual electric array that described strip light pattern forms on chip electrode group plane comprises equidistant array, not equidistant array or the dummy electrodes array of both combinations.
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| CN107843541A (en) * | 2017-10-24 | 2018-03-27 | 上海大学 | A kind of real-time monitoring system and method for unicellular organism physical characteristic |
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| CN101580797B (en) * | 2009-06-19 | 2012-02-22 | 东南大学 | Low-cost cellular positioning arrangement chip and application method |
| CN102580794B (en) * | 2011-01-13 | 2014-03-19 | 博奥生物有限公司 | Micro-fluidic chip capable of positioning cells and organisms and application thereof |
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| CN105602836B (en) * | 2016-02-03 | 2017-10-27 | 河海大学常州校区 | Photo-dielectric swimming assembling chip and the method for building structure gradient Gel Compositions body |
| JP2019062869A (en) * | 2017-10-05 | 2019-04-25 | ソニーセミコンダクタソリューションズ株式会社 | Cell potential detector, method for manufacturing cell potential detector, and information processing system |
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| CN101135680A (en) * | 2007-07-13 | 2008-03-05 | 东南大学 | Light-induction dielectrophoresis auxiliary unicellular dielectric spectrum automatic test equipment and testing method |
| CN201075104Y (en) * | 2007-07-13 | 2008-06-18 | 东南大学 | Device for automatically testing single cell dielectric spectrum based on composite dielectrophoresis |
| CN201188104Y (en) * | 2008-05-13 | 2009-01-28 | 东南大学 | High flux test chip |
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| CN101135680A (en) * | 2007-07-13 | 2008-03-05 | 东南大学 | Light-induction dielectrophoresis auxiliary unicellular dielectric spectrum automatic test equipment and testing method |
| CN201075104Y (en) * | 2007-07-13 | 2008-06-18 | 东南大学 | Device for automatically testing single cell dielectric spectrum based on composite dielectrophoresis |
| CN201188104Y (en) * | 2008-05-13 | 2009-01-28 | 东南大学 | High flux test chip |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107843541A (en) * | 2017-10-24 | 2018-03-27 | 上海大学 | A kind of real-time monitoring system and method for unicellular organism physical characteristic |
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