CN105460882A - Graphene three-dimensional microelectrode array chip, and method and application thereof - Google Patents
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Abstract
The invention relates to a graphene three-dimensional microelectrode array chip, and a method and application thereof. The graphene three-dimensional microelectrode array chip is characterized in that a micro-column array is produced by utilizing negative photoresist, and a microelectrode array is produced by covering a single-layer graphene film on the microelectrode array; and the microelectrode array chip comprises two parts, namely a transparent graphene three-dimensional microelectrode array area and a peripheral gold electrode lead pin. A microelectrode site is a three-dimensional bulge. The three-dimensional microelectrode dome shaped (or hill shaped) microelectrode structure is beneficial for the rigid microelectrode site and the soft cell or tissue to form tight electric coupling, and the graphene has excellent electrical property, so that the electrophysiological detection sensitivity of the microelectrode array can be improved. In addition, the graphene three-dimensional microelectrode array on a transparent substrate is convenient for observation through an inverted microscope and application of various cell microscopic imaging methods, and can be combined with a micro-fluidic chip.
Description
Technical field
The present invention relates to a kind of Graphene three-dimensional micro-electrode array chip, preparation method and application, Graphene three-dimensional micro-electrode array chip provided by the invention can be applicable to large biological molecule, cell or tissue detection field, is especially applied to the electro physiology detection of cell, tissue.
Background technology
Microelectrode array chip utilizes micro fabrication by integrated for the electrode of the multiple micron level a kind of sensor being used for bio signal on one chip and detecting.Microelectrode array chip can detect the electrical signal of large biological molecule in solution simultaneously, can detect the bioelectrical signals of biological cells and tissues, or is used for carrying out electro photoluminescence to biological cells and tissues.
At present, be the metals such as gold, platinum as the electrode material of microelectrode array chip is many, but their opaque character can not widely used inverted microscope in compatible cell biology.When the electrophysiologic activity utilizing such microelectrode array chip to carry out cell or tissue sheet detects, need to use be furnished with immersion type object lens just put microscope, there is operation inconvenience and the sample after being detected can not continue the problems such as cultivation can only abandon.At present based on good electric conductivity and transparency, tin-doped indium oxide (ITO) becomes topmost transparent conductive material, can be used for making microelectrode array chip.But compared with gold, platinum microelectrode, it is high to there is electrochemical impedance in ITO microelectrode, the shortcomings such as electrochemical stability difference in the solution.Developing high performance transparent microelectrode array chip is a current problem urgently to be resolved hurrily.Emerging focus material Graphene has the excellent properties such as high conductivity, high light transmittance, high mechanical properties, good electrochemical stability and good biocompatibility, start to be used to make microelectrode array, and neuronic action potential (XiaoweiDu can be detected, LeiWu, JiCheng, ShanluoHuang, QiCai, QinghuiJin, JianlongZhao, Graphenemicroelectrodearraysforneuralactivitydetection, JBiolPhys (2015) 41:339 – 347).Current, its electrode site of Graphene microelectrode array of plane can not form close contact with the biological cells and tissues of softness, have impact on the sensitivity that electro physiology detects.Nearest theoretical research shows: the form of single-layer graphene on matrix is not subject to the impact of matrix hardness and matrix amplitude change, show as the morphological character (LitingXiong of matrix surface of fitting completely, YuanwenGao.Surfaceroughnessandsizeeffectsonthemorphology ofgrapheneonasubstrate.PhysicaE:Low-dimensionalSystemSys temsandNanostructures.2013,172 (1-2): 154-161).Based on this, the application intends proposing a kind of making Graphene three-dimensional micro-electrode array chip, method and application, to improving the performance of microelectrode array further.
Summary of the invention
In order to the electrode site overcoming existing Graphene microelectrode array chip plane can not form the shortcoming of close contact with the cell or tissue of flexibility, and increase the specific area of electrode site, the object of the present invention is to provide Graphene three-dimensional micro-electrode array chip, method and application thereof, specifically can be applicable to the electrophysiologicalsignal signal detection of biological cells and tissues.
Object of the present invention is reached by following measures: described chip comprises transparent Graphene three-dimensional micro-electrode array region and peripheral gold electrode lead-in wire pin field two parts (Fig. 1); Described chip for substrate, utilizes lift-off technique to make gold electrode lead-in wire and pin (Fig. 2 .a) with silicon, quartz or Pyrex outside microelectrode array region; SU-8 or PI micro-pillar array (Fig. 2 .b is made in microelectrode array region, post height and diameter ratio are about 0.3), again by Graphene/poly-methyl acid methyl esters film transfer to microelectrode array region, go between with the gold electrode that substrate makes in advance and form good Ohmic contact (Fig. 2 .c), then using AZ4620 photoresist as the patterned mask layer of Graphene, etch (Fig. 2 .d) Graphene with oxygen plasma (OxygenPlasma), the Graphene etched comprises the microelectrode site that bends to three-dimensional mound type and two-dimensional electrode and to go between two parts; Finally utilize PI photoresist to make insulating barrier at patterned graphenic surface and gold electrode wire surface, expose Graphene microelectrode site and the gold electrode pin (Fig. 2 .e) of three-dimensional mound type.
The invention has the advantages that can produce simultaneously several to dozens of Graphene three-dimensional micro-electrode array site; Photoresist microtrabeculae has smooth edge, can jack-up graphene film and do not damage graphene film; Biological cells and tissues can wrap the Graphene microelectrode site of mound shape (or be called hills shape, lower with), improves the ability that microelectrode detects ultra-weak electronic signal; The transparent microelectrode array chip utilizing the electrical properties of Graphene excellence, high light transmittance, excellent electrochemical stability and biocompatibility to make is convenient to be combined with inverted microscope, makes many bio-imaging means such as high-resolution fluorescence imaging energy and electro physiology detect coupling.
In sum, the present invention relates to a kind of Graphene three-dimensional micro-electrode array chip, method and application thereof.It is characterized in that the described negative photoresist that utilizes makes micro-pillar array, micro-pillar array covers single-layer graphene film and produces microelectrode array; Described micro-electrode chip comprises transparent Graphene three-dimensional electrode arrays region and peripheral gold electrode lead-in wire pin two parts.Microelectrode site is three-dimensional protruding.The microelectrode site that three-dimensional microelectrode mound shape microelectrode structure is beneficial to rigidity forms electricity closely with the cell or tissue of softness and is coupled, and adds the electrology characteristic of Graphene excellence, can improve the electro physiology detection sensitivity of microelectrode array.In addition, the Graphene three-dimensional micro-electrode array made on a transparent substrate is convenient to observe with inverted microscope, is convenient to the application of various kinds of cell micro imaging method and uses in conjunction with micro-fluidic chip.
Accompanying drawing explanation
Fig. 1 is Graphene three-dimensional micro-electrode array chip schematic diagram of the present invention, a) is chip pictorial diagram, b) is Graphene three-dimensional micro-electrode array region.
Fig. 2 is Graphene three-dimensional micro-electrode array chip fabrication technique flow chart of the present invention, a () makes gold electrode lead-in wire and pin by lift-off technique, b () makes micro-pillar array with SU8 or PI, c () Graphene shifts, d () Graphene is graphical, (e) SU8 or PI makes insulating barrier.
Fig. 3 is mound shape Graphene microelectrode site electrochemical impedance spectroscopy (have chosen three microelectrode site) of the present invention.
Fig. 4 is mound shape Graphene microelectrode site cyclic voltammetry scan curve (have chosen three microelectrode site) of the present invention.
Fig. 5 is that the human neuroblastoma cells (SH-SY5Y) be incubated on Graphene three-dimensional micro-electrode array chip observed by the present invention's inverted microscope, and detects the spontaneous action potentials of cell by multi channel signals detection system.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
Graphene three-dimensional micro-electrode chip fabrication technique flow process is as shown in Figure 2, specific as follows:
(1) substrate is cleaned: use Phiranha solution cleaning silicon chip or quartz plate or Pyrex sheet (as Prex7740), cleaner with deionized water rinsing, nitrogen dries up, oxygen plasma treatment 5 minutes;
(2) Lift-off technique makes outer microelectrode array region external electrode lead-in wire and pin: spin coating AZ4620 on substrate, photoetching process is carried out graphically, and then splash-proofing sputtering metal layer (titanium/gold), removes photoresist with acetone, leave metal layer.(Fig. 2 .a)
(3) three-dimensional micro-pillar array makes: spin coating negative photoresist (silicon chip SU83005, quartz plate and Prex7740 Pyrex PI7510), controls rotating speed 300r/min, after photoetching development, through overcuring, forms micro-pillar array.(Fig. 2 .b)
(4) Graphene transfer: use single-layer graphene film prepared by chemical vapour deposition (CVD), reality is " copper/Graphene/poly-methyl acid methyl esters " composite membrane; This laminated film ammonium persulfate immersion corrosion Copper Foil, after copper is corroded completely, leaves supporting layer " Graphene/poly-methyl acid methyl esters " film; Use rinsed with deionized water film, then by complete film transfer in three-dimensional micro-pillar array, cover array and with gold electrode wire contacts; After leaving standstill a period of time, dry 30 minutes with 85 DEG C of baking ovens, then soak in acetone soln and remove poly-methyl acid methyl esters; Finally clean substrate successively by ethanol, deionized water.(Fig. 2 .c)
(5) make Graphene microelectrode: spin coating AZ4620P photoresist, make Graphene figure by photoetching and oxygen plasma etch, then remove photoresist with acetone, by ethanol, deionized water, substrate is cleaned successively.(Fig. 2 .d)
(6) make electrode dielectric layer: spin coating SU83005, carry out photoetching, development, expose Graphene microelectrode site and gold electrode pin.(Fig. 2 .e)
Embodiment 2
Graphene three-dimensional micro-electrode method for testing performance
Electrochemical impedance and cyclic voltammetry curve method of testing specific as follows:
(1) use electrochemical workstation Gamryreference600, adopt three-electrode system to carry out the electrochemical Characterization of microelectrode.
(2) be bonded in substrate with PDMS by a plastic chamber, microelectrode array region is positioned at cavity.PBS solution is injected in cavity.
(3) using Graphene three-dimensional micro-electrode site as working electrode, Ag/AgCl is as reference electrode, and platinum filament is as to electrode, and three electrodes immerse the PBS solution described in (2).
(4), when carrying out electrochemical analysis, it is the sinusoidal ac signal of 50mV that electrochemical workstation exports peak value, and frequency range is from 0.01HZ to 1MHz.
(5) when carrying out cyclic voltammetric analysis, scanning voltage scope-0.5V-0.5V, sweep speed 100mV/s.
As shown in Figure 3, when 1kHz, it is 300K about Ω that Graphene three-dimensional micro-electrode structure measures less resistance value to electrochemical impedance testing result, can meet the impedance conditions of cell detection.For conventional planar microelectrode structure, after insulating barrier encapsulation, its electrode site can lower than the height of insulating barrier, and electrod-array will be sunk structure one by one.Through three-dimensional mound provided by the invention shape bulge-structure, not only increase the surface area of electrode site, also ensure that cell tissue to be measured contacts with the effective of electrode site simultaneously, avoid because of the charge leakage sternly do not caused of fitting between cell and electrode, improve the signal to noise ratio detected.
Cyclic voltammetry curve test as shown in Figure 4, measures multiple Graphene electrodes, and obtain repeatability CV figure preferably, each all has stable charge transport capability to confirm mound provided by the invention shape Graphene microelectrode site.Though the present embodiment only have chosen three microelectrode site, all applicable to shape electrode site, dozens of mound.
Embodiment 3
Graphene three-dimensional micro-electrode array chip carries out cell chulture and detection
(1) surface treatment of Graphene three-dimensional micro-electrode array: before inoculating cell, Graphene three-dimensional micro-electrode soaks after one hour in the ethanolic solution of 75%, dries under uviol lamp; Then by poly d-lysine (PDL) solution according to 2 μ g/cm
2concentration drip on Graphene three-dimensional micro-electrode array surface, ambient temperature overnight, then use ultrapure water three times, air-dry.
(2) Graphene three-dimensional micro-electrode array surface seeding cell: with digestive juice (0.25% trypsase, 0.02%EDTA solution) by the SH-SY5Y cell dissociation of cellar culture, centrifugal, piping and druming, with 1.0 × 10
7cells/mm
3density be inoculated on Graphene three-dimensional micro-electrode array chip.
(3) SH-SY5Y cell induction differentiation: because ATRA can induce SH-SY5Y to be divided into nerve cell, thus nutrient solution adopt containing 10nM ATRA, 10% hyclone, 0.11g/L Sodium Pyruvate, 300mg/L glutamine MEM/F12 culture medium; Because of vitamin A acid photo-labile, lucifuge in incubation, every day changes liquid.
(4) cell detection: cultivate after 5 days, Graphene three-dimensional micro-electrode array chip is fixed in the fixture of multichannel electrical signal detection system, then fixture is put on the sample stage of inverted microscope, while carrying out imaging, record the spontaneous action potentials that cell produces, testing result as shown in Figure 5.
Claims (9)
1. a Graphene three-dimensional micro-electrode array chip, is characterized in that utilizing negative photoresist to make micro-pillar array, micro-pillar array covers single-layer graphene film and produces microelectrode array; Described micro-electrode chip comprises transparent Graphene three-dimensional electrode arrays region and peripheral gold electrode lead-in wire pin two parts.
2. by chip according to claim 1, it is characterized in that with silicon chip, quartz or Pyrex for substrate graphene film is made into microelectrode array, utilize the negative photoresist microtrabeculae of solidification the Graphene jack-up of two dimension to be formed three-dimensional mound shape microelectrode site, and do not damage graphene film.
3. by chip according to claim 2, when it is characterized in that using silicon chip as substrate, adopt photoresist SU8 to make micro-pillar array, during using quartz and Pyrex as substrate, adopt polyimides photoresist making micro-pillar array; There is the edge of brilliance.
4., by micro-electrode chip according to claim 2, it is characterized in that microelectrode site that mound shape microelectrode array of structures is beneficial to rigidity forms electricity closely with the cell or tissue of softness and is coupled.
5., by the microelectrode array chip described in claim 2 or 4, it is characterized in that described microelectrode site is several to dozens of, for three-dimensional protruding.
6. make the method for the Graphene three-dimensional micro-electrode chip according to any one of claim 1-3, it is characterized in that concrete steps are:
(1) substrate is cleaned: use Phiranha solution cleaning silicon chip, quartz plate or Pyrex sheet, cleaner with deionized water rinsing, nitrogen dries up, oxygen plasma treatment 5 minutes;
(2) make outer microelectrode array region external electrode lead-in wire and pin with Lift-off stripping technology: spin coating AZ4620 in substrate, photoetching process is carried out graphically, and then sputtered titanium/gold metal layer, removes photoresist with acetone, leaves metal layer;
(3) three-dimensional micro-pillar array makes: spin coating negative photoresist, controls rotating speed 250-350r/min, after photoetching development, through overcuring, forms micro-pillar array;
(4) Graphene transfer: use single-layer graphene film prepared by chemical vapour deposition (CVD), reality is " copper/Graphene/poly-methyl acid methyl esters " composite membrane; This laminated film ammonium persulfate immersion corrosion Copper Foil, after copper is corroded completely, leaves supporting layer " Graphene/poly-methyl acid methyl esters " film; Use rinsed with deionized water film again, then by complete film transfer in three-dimensional micro-pillar array, cover array and with gold electrode wire contacts; After leaving standstill a period of time, dry 30 minutes with 85 DEG C of baking ovens, then soak in acetone soln and remove poly-methyl acid methyl esters; Finally clean substrate successively by ethanol, deionized water;
(5) make Graphene microelectrode: spin coating AZ4620P photoresist, make Graphene figure by photoetching and oxygen plasma etch, then remove photoresist with acetone, by ethanol, deionized water, substrate is cleaned successively;
(6) make electrode dielectric layer: spin coating SU83005, carry out photoetching, development, expose Graphene microelectrode site and gold electrode pin.
7., by method according to claim 6, it is characterized in that:
1. described Pyrex sheet Prex7740;
2. silicon chip SU83005 negative photoresist when three-dimensional micro-pillar array makes, quartz plate or Pyrex sheet adopt PI7510;
3. Graphene three-dimensional micro-electrode array is on a transparent substrate convenient to observe with inverted microscope.
8., by the application of Graphene three-dimensional micro-electrode array chip according to claim 1, it is characterized in that the result of the cultivation and detection carrying out cell is:
1. described Graphene three-diemsnional electrode site structure, the resistance value when 1KHz is 300k Ω, meets the impedance of cell detection;
2. described shape Graphene electrodes site, mound each there is stable charge transport capability.
9., by application according to claim 8, it is characterized in that concrete steps are:
(1) surface treatment of Graphene three-dimensional micro-electrode array chip: before inoculating cell, Graphene three-dimensional micro-electrode soaks after one hour in the ethanolic solution of 75%, dries under uviol lamp; Then by poly d-lysine PDL solution according to 2 μ g/cm
2concentration drip on Graphene three-dimensional micro-electrode array surface, ambient temperature overnight, then use ultrapure water three times, air-dry;
(2) Graphene three-dimensional micro-electrode array surface seeding cell: with the digestive juice of 0.25% trypsase and 0.02%EDTA solution composition by the SH-SY5Y cell dissociation of cellar culture, centrifugal, piping and druming, with 1.0 × 10
7cells/mm
3density be inoculated on Graphene three-dimensional micro-electrode array chip;
(3) SH-SY5Y cell induction differentiation: because ATRA can induce SH-SY5Y to be divided into nerve cell, thus nutrient solution adopt containing 10nM ATRA, 10% hyclone, 0.11g/L Sodium Pyruvate, 300mg/L glutamine MEM/F12 culture medium; Because of vitamin A acid photo-labile, lucifuge in incubation, every day changes liquid;
(4) cell detection: cultivate after 5 days, Graphene three-dimensional micro-electrode array chip is fixed in the fixture of multichannel electrical signal detection system, then fixture is put on the sample stage of inverted microscope, while carrying out imaging, records the spontaneous action potentials that cell produces.
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| CN107475072A (en) * | 2017-08-24 | 2017-12-15 | 浙江理工大学 | Cell dynamic characteristic monitoring system and method based on three-dimensional grapheme Interfacial electrode |
| CN109468220A (en) * | 2018-11-15 | 2019-03-15 | 江南大学 | Graphene chip for microbial cell biological information real-time detection |
| CN109468220B (en) * | 2018-11-15 | 2020-05-01 | 江南大学 | Graphene chip for real-time detection of biological information of microbial cells |
| CN109655084A (en) * | 2018-12-10 | 2019-04-19 | 上海交通大学 | It is a kind of can driving neural photoelectrode array preparation method |
| CN113061524A (en) * | 2020-06-17 | 2021-07-02 | 山东大学 | Double-layer micro-fluidic chip, breast cancer miRNA detection kit containing same, preparation method and detection method |
| CN113061524B (en) * | 2020-06-17 | 2024-01-02 | 山东大学 | Double-layer microfluidic chip and breast cancer miRNA detection kit |
| CN114632559A (en) * | 2022-01-26 | 2022-06-17 | 浙江大学 | On-chip micro-groove array digital PCR chip based on electrical impedance detection and manufacturing method thereof |
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