CN104820004A - Cell impedance sensor based on DFP-ITO electrode and application of cell impedance sensor - Google Patents
Cell impedance sensor based on DFP-ITO electrode and application of cell impedance sensor Download PDFInfo
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Abstract
The invention belongs to the technical field of cell impedance sensors and particularly relates to a cell impedance sensor based on a DFP-ITO electrode and application of the cell impedance sensor. The DFP-ITO electrode comprises a conductive substrate layer and an insulating layer located on the conductive substrate layer, wherein the insulating layer is provided with electrode holes, the conductive substrate layer is further provided with lead ports, the conductive substrate layer is indium tin oxide conductive glass, and the insulating layer is a light-sensing dry film. The cell impedance sensor comprises the DFP-ITO electron and a measuring cuvette. The cell impedance sensor is simple in structure, easy to machine, capable of acquiring the morphology and impedance feature values of cells on the DFP-ITO electrode at the same time, capable of being popularized in common labs, and applicable to research fields such as cell physiology and pathology behaviors and drug screening.
Description
Technical field
The invention belongs to electric cell-substrate impedance sensor technical field, be specifically related to a kind of electric cell-substrate impedance sensor based on DFP-ITO electrode and application thereof, detect while described application is specifically related to utilize this electric cell-substrate impedance sensor to realize cytomorphology and impedance information.
Background technology
Electric cell-substrate impedance sensor, as a kind ofly having continuity, can detecting biological behaviours such as cell adherence, propagation, apoptosis without the cell in vitro analytical technology of aggressive feature, obtains extensive Study and appliance in the fields such as drug screening, poisonous substance test, cell physiological parameters analysis and research.
Electric cell-substrate impedance sensor technology is at first by reports such as Giaever and Keese, the cell impedance biosensor of this technique construction is built by the two-electrode system of a pair gold medal material, comprise working electrode that an area is 250 μm of diameters and one large-area to electrode.When cell changes at electrode surface genetic morphology, cell is as the electricity component with frequency dependence, its existence causes the impedance spectrum of system to change, biological behaviour corresponding to cell is obtained by resolving system impedance spectrum, this technology is called as electronic cell-substrate impedance sensing technology (electric cell-substrate impedance sensing, ECIS).In electric cell-substrate impedance sensor research, gold electrode have excellent electric conductivity, biocompatibility and be widely used in detecting cell adherence, propagation, tumour cell mesenchymal transformation, cell is to information such as medicine responses, its elementary tactics indirectly obtains cellular informatics by measuring the impedance data modal data that obtains with theoretical Equivalent Circuit Fitting, has the advantage without invasive, continuity detection.But using Precious Metals-Gold as electrode material, not only cost is high, and light transmission is poor, limits the observation of cytomorphology on gold copper-base alloy electrode.And in fact, the morphological data of cell is the important cell behaviors information of a class, obtained the information such as propagation, apoptosis, subcellular fraction micromechanism, expression of cellular proteins of cell by morphological data.Phase specific impedance data, morphological data is more directly perceived, be more readily understood.Therefore, build a kind of can obtain cell simultaneously impedance information and morphologic cell impedance biosensor will contribute to improving accuracy and the multifunctionality of biology sensor.
Indium tin oxide (ITO) has the conductive material of good light permeability, biocompatibility, therefore can be used as a kind of electrode material and builds various cell biological sensor.ITO conducting film sputters at glass surface by plane magnetic control technology, has even, the good transmittance of surface resistance and thermal stability, market can obtain the commercialization ITO electro-conductive glass of different conductance.ITO can keep potential stability in neutral electrolyte solution, therefore can be used as the electrology characteristic that electrode material detects the cell in culture environment, the light transmission that simultaneously ITO electrode material is good is integrated with optical microscope, by the morphologic information of cell in Digital image technology acquisition ITO electrode.In addition, report for work in prior art and be used for the ITO microelectrode that polyorganosiloxane resin applies to record the application of neuronal cell spike potential, and by the application as endothelial cell impedance biosensor of photoetching technique and the transparent ITO silicon nitride electrode of reactive ion etching technique processing optical, and research finds, ITO electrode material has more excellent light transmission than gold electrode, can detect the respondent behavior of cytochalasin D to porcine pulmonary artery endothelial cell.Although above-mentioned research reports the using value of ITO electrode material at cell impedance biosensor, ITO electrode processing need rely on expensive sputtering and lithographic equipment, and complex process, limits ITO electrode in the preparation of cell impedance biosensor and popularization.
For these reasons with existing technology, still need exploitation further and optimize ITO electrode and preparation method thereof, make that its technique is simple, cost of manufacture reduces, be widely used in cell impedance biosensor, and realize cytomorphology and impedance information detects simultaneously.
Summary of the invention
Photosensitive dry film is mainly used in plated circuit plate technique as a kind of photopolymer material.Relative to liquid photoresist, photosensitive dry film has many advantages, as good adaptability, and substrate adherence, planarization, photosensitive material even distribution, low exposure power consumption and low cost.What is more important, photosensitive dry film photoetching does not need ultra-clean space and expensive lithographic equipment, and the substitute as liquid photoresist also can be applicable to microfluidic channel processing, electroforming mould etc.Based on the above-mentioned characteristic of photosensitive dry film, present inventor finds photosensitive dry film to be applied to ITO electrode processing, does not only need to etch ITO, reduces process complexity, but also reduce the contact resistance of ITO electrode, and the ITO electrode that minimum diameter is 100 μm can be obtained.The binary channels that photosensitive dry film-ITO (DFP-ITO) electrode adopting photosensitive dry film to prepare can realize cytomorphology and impedance information as electric cell-substrate impedance sensor obtains.The present invention completes based on the above-mentioned discovery of inventor just.
Therefore, the present invention provide firstly a kind of photosensitive dry film-indium-tin oxide electrode and preparation method thereof, and this electrode light transmission is good, and its preparation method is simple; Two of object of the present invention is to provide a kind of electric cell-substrate impedance sensor based on photosensitive dry film-indium tin oxide (DFP-ITO) electrode, and this electric cell-substrate impedance sensor structure is simple, and the binary channels that can realize cytomorphology and impedance information obtains; Three of object of the present invention is to provide the application of the described electric cell-substrate impedance sensor based on DFP-ITO electrode, and the method detected while being specifically related to utilize this electric cell-substrate impedance sensor to realize cytomorphology and impedance information, the method is simple to operate.
For achieving the above object, technical scheme of the present invention is:
Photosensitive dry film-indium-tin oxide electrode, is comprised conductive basal layer and is positioned at the insulation course on described conductive basal layer, described insulation course is provided with electrode hole, described conductive basal layer is also provided with pin interfaces, is connected by pin interfaces with power supply; Described conductive basal layer is indium tin oxide electro-conductive glass, and described insulation course is photosensitive dry film.
Indium tin oxide (ITO) has the conductive material of good light permeability, biocompatibility, ITO conducting film sputters at glass surface by plane magnetic control technology, there is even, the good transmittance of surface resistance and thermal stability, market can obtain the commercialization ITO electro-conductive glass of different conductance.ITO can keep potential stability in neutral electrolyte solution, can be used as the electrology characteristic that electrode material detects the cell in culture environment, the light transmission that simultaneously ITO electrode material is good is integrated with optical microscope, by the morphologic information of cell in Digital image technology acquisition ITO electrode.Therefore, ITO electrode has the potentiality realizing cell impedance spectrum information and morphologic information and detect simultaneously.
For realizing the information acquisition of cell impedance spectrum, need processing ITO working electrode.In prior art, processing ITO electrode mainly adopts two class methods: a class is top-down approach, namely on ITO plate, processes ITO electrode by chemical etching; Another kind of is bottom-up approach (approach), namely in patterned substrate, processes ITO electrode by sputtering mode.But the ITO electrode contact resistance that etching and sputtering are formed is comparatively large, causes electric cell-substrate impedance sensor sensitivity decrease; In addition, etching and sputtering approach processing ITO electrode adopt liquid photoresist as electrode image sacrifice layer, and based on photoetching technique, processing technology, cost are higher.
Photosensitive dry film is a kind of photopolymer based on acrylate, there is the features such as such as good adaptability, substrate adherence, planarization, photosensitive material even distribution, low exposure power consumption and low cost, except being applied in PCB technology, also report is had, as micro-fluidic chip in other RESEARCH ON CELL-BIOLOGY field.But the cell biological compatibility of photosensitive dry film but has no report.Present inventor has investigated photosensitive dry film cell biological compatibility from cell adherence and cytotoxicity, result showed cell can adhere at photosensitive dry film and sprawl, Rhodamine 123 has good biologically active to the cell that cell mitochondrial fluorescent dye display sticks to photosensitive dry film surface, and the growth interface that photosensitive dry film can be cell and provides suitable is described.Further, experiment finds, the photosensitive dry film cytotoxicity without step of exposure is significantly higher than the photosensitive dry film through exposure-processed, and extends with incubation time, and cytotoxicity efficiency is more obvious.
The employing ITO microelectrode of existing bibliographical information is built processing technology and parameter that cell impedance biosensor relates to by present inventor and photosensitive dry film of the present invention-indium-tin oxide electrode processing technology compares, the results are shown in Table 1, of the present invention using photosensitive dry film as insulation course, with commercialization ITO electro-conductive glass for substrate, by carrying out selectivity ultraviolet irradiation to photosensitive dry film, the method that development obtains ITO electrode does not need to etch ITO, reduce the contact resistance of process complexity and ITO electrode, and the ITO electrode that minimum diameter is 100 μm can be obtained.
Based on the electric cell-substrate impedance sensor of photosensitive dry film-indium-tin oxide electrode, comprise photosensitive dry film-indium-tin oxide electrode and measure little Chi, described measurement little Chi is placed on the electrode hole of photosensitive dry film-indium-tin oxide electrode, and is communicated with electrode hole.The electric cell-substrate impedance sensor built based on DFP-ITO electrode can coupling optical micro-imaging technique and electrochemical impedance spectral technology, detects while realizing cytomorphology and impedance information.The cytomorphology data that same electrode obtains and impedance information complement one another, verify, improve the comprehensive and accuracy that cellular informatics detects.
Further, described electric cell-substrate impedance sensor, is also provided with packing ring between described measurement little Chi and electrode hole.Packing ring can be falope ring, rubber ring etc., plays anti-skidding and anti-nutrient solution seepage.
Further, described electric cell-substrate impedance sensor, also comprises the fixture of fixing photosensitive dry film-indium-tin oxide electrode and measurement little Chi.For ease of cell chulture and electrochemical impedance spectrometry, DFP-ITO electrode and measurement little Chi are fixed by fixture.
Further, described electric cell-substrate impedance sensor, described fixture comprises train wheel bridge and lower plate, and described train wheel bridge and lower plate are bolted.
Further, described fixture can be polymethylmethacrylate (PMMA) fixture.
Further, described electric cell-substrate impedance sensor, the diameter of the electrode hole of described photosensitive dry film-indium-tin oxide electrode is 0.1mm-2mm.
The preparation method of photosensitive dry film-indium-tin oxide electrode of the present invention, described preparation method is by carrying out selectivity ultraviolet irradiation, development acquisition electrode hole to the photosensitive dry film insulation course be positioned on described conductive basal layer, thus obtains photosensitive dry film-indium-tin oxide electrode.Do not need to etch ITO by the method for photosensitive dry film being carried out to selectivity ultraviolet irradiation, development obtains ITO electrode, reduce the contact resistance of process complexity and ITO electrode, and the ITO electrode that minimum diameter is 100 μm can be obtained.
Further, described preparation method, adopts sodium carbonate liquor to develop; By the photosensitive dry film-indium-tin oxide electrode deionized water rinsing obtained, dry, then through ultraviolet irradiation, photosensitive dry film is solidified completely.
Further, described preparation method specifically comprises the step of carrying out as follows:
(1) overlayed by photosensitive dry film at indium tin oxide conductive glass surface, indium tin oxide electro-conductive glass is conductive basal layer, and photosensitive dry film is insulation course;
(2) draw electrode hole and pin interfaces pattern, on transparent film, make photomask by printing of inkjet printer, through photomask, ultraviolet irradiation is carried out to photosensitive dry film;
(3) the postradiation photosensitive dry film of step (2) medium ultraviolet is developed, the region without ultraviolet irradiation is exposed, and then obtain photosensitive dry film-indium-tin oxide electrode.
Electric cell-substrate impedance sensor of the present invention can obtain impedance information and the morphologic information of cell simultaneously, therefore, present invention also offers and carry out based on described electric cell-substrate impedance sensor the method that cytomorphology and impedance information detect simultaneously, comprise the step of carrying out as follows:
(1) cell suspension is loaded in measurement little Chi, leaves standstill and make cell settlement to the surface of electrode hole, put into incubator and cultivate;
(2) by cytomorphology change information in optical microphotograph imaging technique observation of cell incubation;
(3) by cell impedance information in electrochemical impedance spectroscopy commercial measurement cell cultivation process, described electrochemical impedance spectroscopy commercial measurement is that the photosensitive dry film-indium-tin oxide electrode being vaccinated with cell carries out electrochemical impedance spectrometry as working electrode.
Beneficial effect of the present invention: (1) photosensitive dry film of the present invention-indium-tin oxide electrode light transmission is good, adopt photosensitive dry film as insulation course, not only do not need to etch ITO, reduce process complexity, but also reduce the contact resistance of ITO electrode, and the ITO electrode that minimum diameter is 100 μm can be obtained, namely reduce ITO electrode difficulty of processing, cost and time, and photosensitive dry film surface suitable cell adherence, cell growth is without obvious cellulotoxic side effect; Photosensitive dry film-indium-tin oxide electrode structure is simple, be easy to processing.(2) based on the electric cell-substrate impedance sensor of photosensitive dry film-indium tin oxide (DFP-ITO) electrode, structure is simple, be easy to processing, can coupling optical micro-imaging technique and electrochemical impedance spectral technology, detect while realizing cytomorphology and impedance information, the cytomorphology data that same electrode obtains and impedance information complement one another, verify, improve the comprehensive and accuracy that cellular informatics detects.(3) method detected while the present invention utilizes this electric cell-substrate impedance sensor to realize cytomorphology and impedance information is simple to operate, can in common lab popularization and for research fields such as the behavior of cell physiological pathology, drug screenings.
Accompanying drawing explanation
The structural representation of Fig. 1 photosensitive dry film-indium-tin oxide electrode.
DFP-ITO electrode machining schematic flow sheet described in Fig. 2 embodiment 2, wherein (a): the ITO electro-conductive glass after cleaning; (b): photosensitive dry film is laminated to ITO conductive glass surface; (c): ultraviolet irradiation is carried out to photosensitive dry film through photomask; D (): dry film develops, obtains DFP-ITO electrode.
Fig. 3 is based on the structural representation of the electric cell-substrate impedance sensor of photosensitive dry film-indium-tin oxide electrode.
The structural drawing of the electric cell-substrate impedance sensor based on photosensitive dry film-indium-tin oxide electrode shown in Fig. 4 embodiment 3, comprise DFP-ITO electrode and electrochemical impedance spectrometry little Chi, measure little Chi containing four independent measuring units, wherein (a): electric cell-substrate impedance sensor assembling schematic diagram; (b): electric cell-substrate impedance sensor parts pictorial diagram; C electric cell-substrate impedance sensor pictorial diagram that () assembles.
The cell biological compatibility testing result of Fig. 5 photosensitive dry film, wherein (a): A549 cell cultivates the light field after 24h and Rhodamine 123 fluorescent dye displaing micro picture on photosensitive dry film surface; (b): post-exposure process is on the Cytotoxic impact of photosensitive dry film A549 (n=5); In figure, * represents p<0.05, and △ represents p<0.01.
Fig. 6 different-diameter DFP-ITO electrode picture and impedance spectrum feature, wherein (a): diameter is respectively the photosensitive dry film ITO picture in kind of 0.5mm, 1.0mm, 1.5mm and 2.0mm; (b): diameter is respectively the photosensitive dry film ITO electrode direct impedance modal data of 1.0mm, 1.5mm and 2.0mm, and electrochemical impedance spectrometry is carried out in 0.01M PBS solution.
Fig. 7 utilizes the morphologic information of A549 cell respectively when 2h, 24h and 48h of observation by light microscope DFP-ITO electrode surface.
On Fig. 8 DFP-ITO electrode, cell adherence propagation behavior electrochemical impedance spectroscopy detects, wherein (a): the electrochemical impedance spectroscopy complex plane figure of A549 cell respectively when 0h, 2h, 24h and 48h being seeded in DFP-ITO electrode surface; (b): biological cell equivalent-circuit model; (c): to the curve-fitting results (n=4) being seeded in the not cell equivalent-circuit model in the same time of A549 cell on DFP-ITO electrode.
Embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, usually conveniently condition.
Experiment material involved in following examples:
ITO electro-conductive glass is purchased from Zhuhai Kaivo Optoelectronic Technology Co., Ltd. (China), and ITO conduction thickness 2200 ± 300 dust, surface resistance≤7 Ω/square, substrate of glass thickness is 1.1mm.
Photosensitive dry film model is HQ-6100, is purchased from Changxing Chemical Industry Co Ltd, and photographic layer thickness is 35um, is a kind of low cost negative photoresist.
A549 cell chulture: people's lung cancer epithelial cell A549 is by centralab of Yongchuan Hospital Affiliated to Chongqing Medical University cellar culture.Cell with containing 10% hyclone (Hangzhou Chinese holly), 100U/ ml penicillin and 100 μ g/ml streptomysins DMEM nutrient solution (Gibco) at 37 DEG C, cultivate under 5% carbon dioxide conditions.Treat that Growth of Cells is to exponential phase, 0.25% pancreatin (containing 0.02%EDTA) vitellophag, the centrifugal 4min of 1500rmp, goes down to posterity by 1:3.A549 passage number of times used herein was 4 to 10 generations, during use, A549 cell DMEM cell culture fluid was made the cell suspension of variable concentrations.
Embodiment 1 photosensitive dry film cell compatibility detects
The cell biological compatibility of photosensitive dry film adopts cell adhesion experiments and cytotoxicity experiment to verify.
(1) cell adhesion experiments flow process: photosensitive dry film being cut into area is 8mm × 8mm square piece, after ultraviolet irradiation, development treatment being carried out to photosensitive dry film by DFP-ITO electrode machining flow process of the present invention, be positioned in 96 orifice plates, 70% (v/v) concentration ethanol is sterilized.By 100 μ l A549 cells with concentration for 2.5 × 10
5cells/ml is seeded in photosensitive dry film surface, 37 DEG C, cultivate 24h under 5% concentration carbon dioxide conditions and make cell attachment, by the PBS solution that concentration is 10 μMs of Rhodamine 123s (Sigma), fluorescent dye is carried out to cell mitochondrial, inverted fluorescence microscope (IX-71, Olympus) observes photosensitive dry film cytomorphology adherent on the surface and cell fluorescent images.
(2) cytotoxicity experiment flow process: by 100 μ l A549 cells with concentration for 5 × 10
4cells/ml density is seeded in 96 orifice plates, hatches 2h and make cell attachment in cell culture incubator.Subsequently photosensitive dry film is immersed in cell culture fluid as experimental group, simultaneously not contain the cell hole of photosensitive dry film for negative control group, only containing the not celliferous hole of photosensitive dry film is blank group, often group establishes 5 multiple holes, cultivate 24h respectively, 48h, after 72h hour, photosensitive dry film is taken out from hole, and in every hole, add 10 μ l Cell CountingKit-8 (CCK-8) analytic liquids (green skies biotech company), jog mixes, 37%, 2h is hatched in 5% carbon dioxide cell incubator, multi-functional microplate reader (Varioskan, Thermo Scientific) measure the absorbance of solution at 450nm.Photosensitive dry film cytotoxicity is defined as: cytotoxicity (%)=1-(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) * 100%.
The present embodiment has investigated photosensitive dry film cell biological compatibility from cell adherence and cytotoxicity, and experimental result as shown in Figure 5.Fig. 5 (a) for A549 cell be seeded in photosensitive dry film 24h cell occur adhere to light field and Fluorescent micrograph, result showed cell can adhere at photosensitive dry film and sprawl, Rhodamine 123 sticks to photosensitive dry film surface A 549 cell to cell mitochondrial fluorescent dye display and has good biologically active, and this result explanation photosensitive dry film can be the growth interface that A549 cell provides suitable.Fig. 5 (b) is the cytotoxicity result of the photosensitive dry film of two kinds of different disposal.Experiment finds, the photosensitive dry film cytotoxicity without post-exposure step is significantly higher than the photosensitive dry film through post-exposure process, and extends with incubation time, and cytotoxicity efficiency is more obvious, and between the cytotoxicity of hatching 72h two kinds of photosensitive dry films, ratio is about 4.86.This result illustrates that the post-exposure of photosensitive dry film affects its Cytotoxic key factor, reason is that post-exposure step can make light sensitive molecule be polymerized completely, avoids unpolymerized photosensitive molecular to be discharged into cell culture fluid when cell chulture and produces cytotoxicity to cell.Meanwhile, start after experiment also finds to soak 24h without the photosensitive dry film of post-exposure process in nutrient solution to occur that softening, surface starts gauffer, then the photosensitive dry film form of exposure-processed does not change.In fact, the photosensitive dry film of post-exposure process still has slight cytotoxicity (72h is 12.6%), infers that reason is that photosensitive dry film has adsorbed protein molecular cell growth in nutrient solution and causes slight suppression.Therefore, with blank nutrient solution, preincubate is carried out to photosensitive dry film before cell inoculation and reduce its cell growth depression effect.
The processing of embodiment 2 photosensitive dry films-indium-tin oxide electrode
ITO electro-conductive glass is used respectively acetone, absolute ethyl alcohol, each 15min of deionized water ultrasonic cleaning, dry stand-by.
The structural representation of the DFP-ITO electrode prepared by the present embodiment as shown in Figure 1.Photosensitive dry film-indium-tin oxide electrode, the insulation course 2 comprising conductive basal layer 1 and be positioned on described conductive basal layer 1, described insulation course 2 is provided with electrode hole 3, and described electrode hole 3 is four, described conductive basal layer 1 is also provided with pin interfaces 4, is connected with power supply by pin interfaces 4; Described conductive basal layer 1 is indium tin oxide electro-conductive glass, and described insulation course 2 is photosensitive dry film.
DFP-ITO electrode machining flow process as shown in Figure 2, specifically comprises the following steps:
(1) photosensitive dry film is overlayed on electro-conductive glass ito film surface by office laminating machine (100 DEG C), and range estimation photosensitive dry film and ito film closely attach and bubble-free between the two;
(2) Visio Coreldraw 12.0 (Corel company, Canada) ITO electrode hole and pin interfaces pattern is drawn out, by printing of inkjet printer (ESPON 1390, Japan) on transparent film, make photomask with 2880dpi resolution printing, through photomask, ultraviolet irradiation 30s is carried out to photosensitive dry film;
(3) by postradiation photosensitive dry film at 30 DEG C with the sodium carbonate development 5min of 1%, the ITO region without ultraviolet irradiation is exposed, acquisition structural representation DFP-ITO electrode as shown in Figure 1.Subsequently by electrode deionized water rinsing 2 times, 60 DEG C of oven dry, ultraviolet irradiation 60s makes photoresists solidify completely.
Embodiment 3 is based on the electric cell-substrate impedance sensor of DFP-ITO electrode
The structural representation of the electric cell-substrate impedance sensor based on DFP-ITO electrode prepared by the present embodiment as shown in Figure 3.
For ease of cell chulture and electrochemical impedance spectrometry, photosensitive dry film-indium-tin oxide electrode prepared by embodiment 2 and measurement little Chi 5 are fixed by polymethylmethacrylate (PMMA) fixture, DFP-ITO electrode is placed in and measures bottom little Chi, measuring little Chi volume is 500 μ L.DFP-ITO electrode, measurement little Chi assembling schematic diagram and pictorial diagram are as shown in Figure 4.
Based on the electric cell-substrate impedance sensor of photosensitive dry film-indium-tin oxide electrode, comprise the photosensitive dry film-indium-tin oxide electrode prepared by embodiment 2 and measure little Chi 5, described measurement little Chi 5 is placed on the electrode hole 3 of photosensitive dry film-indium-tin oxide electrode, and be communicated with electrode hole 3, packing ring 6 is also provided with between described measurement little Chi 5 and electrode hole 3, packing ring 6 is falope ring, also comprise the fixture 7 of fixing photosensitive dry film-indium-tin oxide electrode and measurement little Chi 5, described fixture 7 comprises train wheel bridge 71 and lower plate 72, described train wheel bridge 71 is connected by bolt 8 with lower plate 72, described fixture 7 material is polymethylmethacrylate (PMMA).
The electric cell-substrate impedance sensor built based on DFP-ITO electrode can coupling optical micro-imaging technique and electrochemical impedance spectral technology, detects while realizing cytomorphology and impedance information.The cytomorphology data that same electrode obtains and impedance information complement one another, verify, improve the comprehensive and accuracy that cellular informatics detects.
Embodiment 4 cell impedance biosensor of the present invention builds cell impedance biosensor with the ITO electrode that prior art is reported and compares
Processing ITO electrode mainly adopts two class methods: a class is top-down approach, namely on ITO plate, processes ITO electrode by chemical etching; Another kind of is bottom-up approach (approach), namely in patterned substrate, processes ITO electrode by sputtering mode.But the ITO electrode contact resistance that etching and sputtering are formed is comparatively large, causes electric cell-substrate impedance sensor sensitivity decrease; In addition, etching and sputtering approach processing ITO electrode adopt liquid photoresist as electrode image sacrifice layer, and based on photoetching technique, processing technology, cost are higher.In the present embodiment, the ITO electrode that the cell impedance biosensor prepare the present invention and prior art are reported builds processing technology that cell impedance biosensor relates to and parameter compares, result is as shown in table 1, result shows, the present invention is using photosensitive dry film as insulation course, with commercialization ITO electro-conductive glass for substrate, by carrying out selectivity ultraviolet irradiation to photosensitive dry film, the method that development obtains ITO electrode does not need to etch ITO, reduce the contact resistance of process complexity and ITO electrode, and the ITO electrode that minimum diameter is 100 μm can be obtained.
Table 1 ITO electrode builds cell impedance biosensor and compares
Embodiment 5 cell impedance biosensor working electrode area is on the research of the impact of electric cell-substrate impedance sensor sensitivity
Build cell impedance biosensor to need to consider a series of important parameter, wherein working electrode area affects one of electric cell-substrate impedance sensor sensitivity important parameter.In the present embodiment, first machined the DFP-ITO electrode of 0.5mm, 1.0mm, 1.5mm and 2mm tetra-kinds of different-diameters (i.e. the diameter of electrode hole 3), have studied the impact of the DFP-ITO electrode pair A549 electric cell-substrate impedance sensor sensitivity of different-diameter, research method is as follows:
Before cell is loaded into DFP-ITO electrode, with 70% ethanol to measurement little Chi sterilization 5min, measure little Chi 2 times by sterilizing washed with de-ionized water subsequently; By 200 μ l A549 cell suspensions with concentration 2.5 × 10
5cells/ml is loaded in the measurement little Chi containing the DFP-ITO electrode of different-diameter respectively, be placed in super-clean bench and under room temperature condition, after static 30min, make cell uniform settling to electrode surface, put into 37 DEG C subsequently, cultivate in 5% carbon dioxide cell incubator, on DFP-ITO electrode, A549 cell impedance information is measured by electrochemical impedance spectroscopy (Electrochemical Impedance Spectroscopy, EIS) technology.Electrochemical impedance spectrometry have employed CS315 electrochemical workstation (Wuhan Koster Instrument Ltd.), electrochemical impedance spectral measurement system adopts three-electrode system, to be vaccinated with the DFP-ITO electrode of cell as working electrode, by Ag/AgCl silk contrast electrode (diameter: 0.5mm, length: 10mm) and platinum filament insert to measure in little Chi to electrode (diameter: 1.0mm, length: 10mm) and carry out electrochemical impedance spectrometry.Measuring using 0.01M PBS as supporting electrolyte solution, take amplitude as 10mV sine wave is pumping signal, and range of scanned frequencies is 1-10
5hz, impedance spectrum measurement data ZView 2.0 software is analyzed.Result as shown in Figure 6.
As shown in Fig. 6 (a), experiment finds, in supporting electrolyte solution, too high open circuit potential (open circuit potential) is there is between 0.5mm diameter photosensitive dry film ITO electrode and contrast electrode, DFP-ITO electrode and the electrolyte solution contacts impedance of prompting 0.5mm diameter are large, infer that its reason is relevant with hydrophobic property with photosensitive dry film thickness.Fig. 6 (b) is depicted as the DFP-ITO electrode impedance spectrum that diameter is respectively 1.0mm, 1.5mm, 2.0mm, between display different-diameter electrode, there is similar frequencydependence characteristic, in its whole frequency range, electrode impedance value becomes inverse relationship with diameter, and its reason is that ITO electrode is relevant with the electric charge bilayer that electrolyte dissolution interface is formed.This result is similar to the gold electrode result of bibliographical information.When cell is in ITO electrode surface adhesion and when sprawling, the ITO electrode little due to area reveals higher sensitivity to cytomorphology change list.
Embodiment 6 utilizes the electric cell-substrate impedance sensor based on DFP-ITO electrode to detect A549 cytomorphology and electrochemical impedance information simultaneously
The feature that ITO builds cell impedance biosensor most attractive force as electrode material is the Dual channel detection that can realize cytomorphology and impedance information.The present embodiment is verified the electric cell-substrate impedance sensor based on DFP-ITO electrode of the present invention, utilizes this electric cell-substrate impedance sensor to have detected cytomorphology and impedance information in the adhesion of A549 cell, breeding simultaneously.
The electric cell-substrate impedance sensor that the present embodiment adopts selects diameter to be 1.0mm ITO electrode.
Detect by the following method:
Before cell is loaded into DFP-ITO electrode, with 70% ethanol, measurement little Chi 5 is sterilized 5min, measure little Chi by sterilizing washed with de-ionized water subsequently 5 twice; By 200 μ l A549 cell suspensions with concentration 2.5 × 10
5cells/ml is loaded in the measurement little Chi 5 containing DFP-ITO electrode, be placed in super-clean bench and under room temperature condition, after static 30min, make cell uniform settling to electrode hole 3 surface, put into 37 DEG C subsequently, cultivate in 5% carbon dioxide cell incubator, DFP-ITO electrode is taken out to be placed on inverted fluorescence microscope objective table respectively after 2h, 24h, 48 and carry out morphological observation and Image Acquisition.Result as shown in Figure 7, A549 cell is seeded in morphology displaing micro picture when 2h, 24h and 48h in ITO electrode, display A549 cell uniform spreading in ITO electrode, sprawl (2h), propagation (24h) and the cell monolayer that can be observed cell by microscope form (48h).This structure illustrates that ITO adheres to and propagation as electrode material suitable cell, and its good light transmission is convenient to obtain cytomorphology data.
On DFP-ITO electrode, A549 cell impedance information is measured by electrochemical impedance spectroscopy (ElectrochemicalImpedance Spectroscopy, EIS) technology.When A549 cell is when DFP-ITO electrode surface adheres to and breed, cell causes system impedance to be composed as the electricity original paper with frequency dependence changing, therefore detect by impedance spectrum technology the adhesion of cell on DFP-ITO electrode, propagation behavior and resolve.Electrochemical impedance spectrometry have employed CS315 electrochemical workstation (Wuhan Koster Instrument Ltd.), electrochemical impedance spectral measurement system adopts three-electrode system, to be vaccinated with the DFP-ITO electrode of cell as working electrode, by Ag/AgCl silk contrast electrode (diameter: 0.5mm, length: 10mm) and platinum filament insert to measure in little Chi to electrode (diameter: 1.0mm, length: 10mm) and carry out electrochemical impedance spectrometry.Measuring using 0.01M PBS as supporting electrolyte solution, take amplitude as 10mV sine wave is pumping signal, and range of scanned frequencies is 1-10
5hz, impedance spectrum measurement data ZView 2.0 software is analyzed.Result is as shown in 8 (a), and A549 cell is seeded in electrochemical impedance spectroscopy complex plane figure corresponding to 0h, 2h, 24h, 48h on DFP-ITO electrode, and the propagation of visible A549 cell in ITO electrode causes system impedance spectrum HFS to change.The HFS that clearer display A549 cell proliferation causes system impedance to be composed in complex plane figure expanded scale (expanded scales) forms semicircle, and along with the increase of cell proliferation HFS half circular diameter.This result illustrates that on electrode interface, cell proliferation causes ionic charge transfer process speed on electrode interface to slow down, and its reason is relevant with cytoplasma membrane capacitive characteristics.In addition, at low frequency region, the propagation of A549 cell does not produce significant change to system impedance spectrum, its reason is that cell is typical insulator at its electrology characteristic of low frequency part, the gas current of electrode and electrolyte solution carries out shifting mainly through space between cells and not through cell, therefore the low frequency region of impedance spectrum can not reflect that cell behavior changes.
For analyzing the capacitive reactance characteristic of different phase A549 cell on DFP-ITO electrode further, present invention employs document [Karimullah A S, Cumming D R S, Riehle M, etal Development of aconducting polymer cell impedance sensor [J] .Sensors and Actuators B:Chemical, 2013, 176:667-674] the cell equivalent-circuit model reported, as shown in Fig. 8 (b), cell equivalent electrical circuit is composed in series by a RC parallel circuit and a R element, wherein, R's theoretical explanation is the gas resistance of cell-electrode, R
cellbe interpreted as cell-ECM resistance, C
cellbe interpreted as the capacity effect of cytoplasma membrane.By curve fitting technique system impedance composed semi-circular portions and cell equivalent-circuit model carries out matching, A549 cell Different growth phases equivalent-circuit component value is as shown in Fig. 8 (c).As can be known from the results, along with the adhesion of A549 cell at ITO electrode interface and propagation, C
cellvalue slowly increases, and R
cellvalue and R's value increase gradually.C after formation cell monolayer
cellvalue is about 2.8nF, R
cellvalue is about 754 Ω, is about 216 Ω.Meanwhile, when A549 cell is from (2h) after adherent to being formed between cell monolayer (48h), R
cellthe gap that value continues to increase between explanation cell-ECM reduces gradually, and Fusion of Cells degree increases; R's value continues to increase, and points out cell-ITO electrode spacing to reduce, cell adherence strength-enhanced.On the contrary, C
cellbe worth the propagation of A549 cell insensitive to the morphological change forming cell monolayer.In addition, carefully analyze the present embodiment data also to find to change less from each component value between cell inoculation (0h) to cell attachment (2h), illustrate that native system is not enough for the cell behaviors detection sensitivity of this process, its reason is that native system uses ITO electrode area (0.785mm
2) comparatively large, A549 cell is not enough to the impedance spectrum of change system in ITO electrode surface adhesion.Therefore, detection electric cell-substrate impedance sensor detection sensitivity can be improved further by electrode modification under the prerequisite not affecting electrode light transmission.
From above interpretation of result, the electric cell-substrate impedance sensor built based on DFP-ITO electrode can coupling optical micro-imaging technique and electrochemical impedance spectral technology, detects while realizing cytomorphology and impedance information.The cytomorphology data that same electrode obtains and impedance information complement one another, verify, improve the comprehensive and accuracy that cellular informatics detects.
Therefore, DFP-ITO electrode of the present invention, adopts photosensitive dry film to reduce ITO electrode difficulty of processing, cost and time as insulation course, and photosensitive dry film surface suitable cell adherence, cell growth and without obvious cellulotoxic side effect; Electric cell-substrate impedance sensor of the present invention is using ITO as electrode, structure is simple, be easy to processing, can be observed the morphologic information of cell on electrode by optical microscope, utilize electrochemical impedance spectral technology and cell Equivalent Circuit Fitting can detect adhesion and the breeding middle impedance information of cell on DFP-ITO electrode; Namely the electric cell-substrate impedance sensor based on DFP-ITO electrode of the present invention can obtain cytomorphology and impedance characteristic value simultaneously, can in common lab popularization and for research fields such as the behavior of cell physiological pathology, drug screenings.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. photosensitive dry film-indium-tin oxide electrode, it is characterized in that, comprise conductive basal layer (1) and be positioned at the insulation course (2) on described conductive basal layer (1), described insulation course (2) is provided with electrode hole (3), described conductive basal layer (1) is also provided with pin interfaces (4), is connected with power supply by pin interfaces (4); Described conductive basal layer (1) is indium tin oxide electro-conductive glass, and described insulation course (2) is photosensitive dry film.
2. based on the electric cell-substrate impedance sensor of photosensitive dry film-indium-tin oxide electrode according to claim 1, it is characterized in that, comprise photosensitive dry film-indium-tin oxide electrode and measure little Chi (5), described measurement little Chi (5) is placed on the electrode hole (3) of photosensitive dry film-indium-tin oxide electrode, and is communicated with electrode hole (3).
3. electric cell-substrate impedance sensor according to claim 2, is characterized in that, between described measurement little Chi (5) and electrode hole (3), be also provided with packing ring (6).
4. electric cell-substrate impedance sensor according to claim 2, is characterized in that, also comprises the fixture (7) of fixing photosensitive dry film-indium-tin oxide electrode and measurement little Chi (5).
5. electric cell-substrate impedance sensor according to claim 4, it is characterized in that, described fixture (7) comprises train wheel bridge (71) and lower plate (72), and described train wheel bridge (71) is connected by bolt (8) with lower plate (72).
6. electric cell-substrate impedance sensor according to claim 2, is characterized in that, the diameter of the electrode hole (3) of described photosensitive dry film-indium-tin oxide electrode is 0.1mm-2mm.
7. the preparation method of photosensitive dry film-indium-tin oxide electrode according to claim 1, it is characterized in that, described preparation method is by carrying out selectivity ultraviolet irradiation, development acquisition electrode hole (3) to the photosensitive dry film insulation course (2) be positioned on described conductive basal layer (1), thus obtains photosensitive dry film-indium-tin oxide electrode.
8. preparation method according to claim 7, is characterized in that, adopts sodium carbonate liquor to develop; By the photosensitive dry film-indium-tin oxide electrode deionized water rinsing obtained, dry, then through ultraviolet irradiation, photosensitive dry film is solidified completely.
9. preparation method according to claim 7, is characterized in that, described preparation method specifically comprises the step of carrying out as follows:
(1) overlayed by photosensitive dry film at indium tin oxide conductive glass surface, indium tin oxide electro-conductive glass is conductive basal layer (1), and photosensitive dry film is insulation course (2);
(2) draw electrode hole (3) and pin interfaces (4) pattern, on transparent film, make photomask by printing of inkjet printer, through photomask, ultraviolet irradiation is carried out to photosensitive dry film;
(3) the postradiation photosensitive dry film of step (2) medium ultraviolet is developed, the region without ultraviolet irradiation is exposed, and then obtain photosensitive dry film-indium-tin oxide electrode.
10. carry out based on electric cell-substrate impedance sensor according to claim 2 the method that cytomorphology and impedance information detect simultaneously, it is characterized in that, comprise the step of carrying out as follows:
(1) cell suspension is loaded in measurement little Chi (5), leaves standstill and make cell settlement to the surface of electrode hole (3), put into incubator and cultivate;
(2) by cytomorphology change information in optical microphotograph imaging technique observation of cell incubation;
(3) by cell impedance information in electrochemical impedance spectroscopy commercial measurement cell cultivation process, described electrochemical impedance spectroscopy commercial measurement is that the photosensitive dry film-indium-tin oxide electrode being vaccinated with cell carries out electrochemical impedance spectrometry as working electrode.
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Application publication date: 20150805 |