CN105460882B - A kind of graphene three-dimensional micro-electrode array chip, method and its application - Google Patents
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Abstract
The present invention relates to a kind of graphene three-dimensional micro-electrode array chip, method and its application.It is characterized in that described utilization negative photoresist makes micro-pillar array, microelectrode array is produced in micro-pillar array overlying lid single-layer graphene film；Described micro-electrode chip includes transparent graphene three-dimensional electrode arrays region and peripheral gold electrode lead pin two parts.Microelectrode site is three-dimensional raised.Three-dimensional microelectrode mound shape (or being hills shape) microelectrode structure is formed and is closely electrically coupled beneficial to rigid microelectrode site and soft cell or tissue, plus the electrology characteristic that graphene is excellent, it is possible to increase the electro physiology detection sensitivity of microelectrode array.In addition, the graphene three-dimensional micro-electrode array made on a transparent substrate is easy to be observed with inverted microscope, it is easy to the application of various kinds of cell micro imaging method and is used with reference to 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, what the present invention was provided
Graphene three-dimensional micro-electrode array chip can be applied to large biological molecule, cell or tissue detection field, be particularly applied to thin
Born of the same parents, the electro physiology detection of tissue.
Background technology
Microelectrode array chip is to be used on one chip using micro fabrication by the electrode of multiple micron levels is integrated
A kind of sensor detected in bio signal.Microelectrode array chip can detect the electricity letter of large biological molecule in solution simultaneously
Number, the bioelectrical signals of cell and tissue can be detected, or for carrying out electro photoluminescence to cell and tissue.
At present, the electrode material of microelectrode array chip it is many as be the metals such as gold, platinum, but their opaque properties can not
Widely used inverted microscope in compatible cell biology.Cell or tissue piece is carried out using such microelectrode array chip
Electrophysiologic activity detection when, it is necessary to just put microscope using equipped with immersion type object lens, there is operation inconvenience and detected
Sample afterwards can not continue the problems such as culture can only be abandoned.It is currently based on good electric conductivity and transparency, tin-doped indium oxide
(ITO) topmost transparent conductive material is turned into, can be for making microelectrode array chip.But with gold, the micro- electricity of platinum
Pole is compared, and ITO microelectrodes have electrochemical impedance height, the shortcomings of electrochemical stability in the solution is poor.Exploitation is high performance
Transparent microelectrode array chip is a current problem urgently to be resolved hurrily.Emerging focus material graphene possesses highly conductive
Property, high light transmittance, high mechanical properties, the excellent properties such as good electrochemical stability and good biocompatibility, start quilt
For making microelectrode array, and can detect neuron action potential (Xiaowei Du, Lei Wu, Ji Cheng,
Shanluo Huang, Qi Cai, Qinghui Jin, Jianlong Zhao, Graphene microelectrode arrays
For neural activity detection, J Biol Phys (2015) 41:339–347).Currently, the graphene of plane
Microelectrode array its electrode site can not be with soft cell and organizing the formation of close contact, and have impact on that electro physiology detects is sensitive
Degree.Nearest theoretical research shows：Form of the single-layer graphene on matrix be not by the shadow of matrix hardness and matrix amplitude change
Ring, show as morphological character (LitingXiong, the YuanwenGao.Surface roughness of laminating matrix surface completely
and size effects on the morphology of graphene on a substrate.Physica E:Low-
dimensional System Systems and Nanostructures.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 further improve microelectrode
The performance of array.
The content of the invention
In order to overcome the electrode site of existing graphene microelectrode array chip plane can not be with flexible cell or tissue
The shortcoming being in close contact is formed, and increases the specific surface area of electrode site, it is an object of the invention to provide graphene three-dimensional is micro-
Electrode array chip, method and its application, are particularly applicable to the electrophysiologicalsignal signal detection of cell and tissue.
The purpose of the present invention is reached by following measures：Described chip includes transparent graphene three-dimensional micro-electrode battle array
Column region and peripheral gold electrode lead pin field two parts (Fig. 1)；Described chip is using silicon, quartz or Pyrex as base
Bottom, gold electrode lead and pin (Fig. 2 .a) are made outside microelectrode array region using lift-off techniques；Microelectrode array area
SU-8 or PI micro-pillar arrays are made in domain, and (Fig. 2 .b, pillar height and diameter ratio are about 0.3), then by graphene/poly- methyl acid methyl esters
Film is transferred to microelectrode array region, and good Ohmic contact (figure is formed with the gold electrode lead of pre-production in substrate
2.c), it is right with oxygen plasma (Oxygen Plasma) then using AZ4620 photoresists as the patterned mask layer of graphene
Graphene is performed etching (Fig. 2 .d), and the graphene etched includes bending to the microelectrode site and two-dimensional electrode of three-dimensional mound type
Lead two parts；Insulating barrier finally is made in patterned graphenic surface and gold electrode wire surface using PI photoresists, cruelly
Expose the graphene microelectrode site and gold electrode pin (Fig. 2 .e) of three-dimensional mound type.
The advantage of the invention is that can produce several to dozens of graphene three-dimensional micro-electrode array site simultaneously；Photoetching
Glue microtrabeculae has smooth edge, can jack-up graphene film without damage graphene film；Cell and tissue can be wrapped up
Firmly the graphene microelectrode site of mound shape (or being hills shape, similarly hereinafter), improves the ability that microelectrode detects ultra-weak electronic signal；
Utilize the transparent micro- of the excellent electrical properties of graphene, high light transmittance, excellent electrochemical stability and biocompatibility making
Electrode array chip is convenient to be used in combination with inverted microscope so that many bio-imaging means such as high-resolution fluorescence imaging energy
Detect and be combined with electro physiology.
In summary, the present invention relates to a kind of graphene three-dimensional micro-electrode array chip, method and its application.Its feature exists
Micro-pillar array is made in described utilization negative photoresist, microelectrode is produced in micro-pillar array overlying lid single-layer graphene film
Array；Described micro-electrode chip includes transparent graphene three-dimensional electrode arrays region and peripheral gold electrode lead pin two
Point.Microelectrode site is three-dimensional raised.Three-dimensional microelectrode mound shape microelectrode structure be beneficial to rigid microelectrode site with it is soft
Soft cell or tissue, which is formed, to be closely electrically coupled, and adds the excellent electrology characteristic of graphene, it is possible to increase microelectrode array
Electro physiology detection sensitivity.In addition, the graphene three-dimensional micro-electrode array made on a transparent substrate, which is easy to use, is inverted micro-
Mirror is observed, and is easy to the application of various kinds of cell micro imaging method and is used with reference to micro-fluidic chip.
Brief description of the drawings
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
Tie up microelectrode array region.
Fig. 2 is graphene three-dimensional micro-electrode array chip fabrication technique flow chart of the present invention, and (a) uses lift-off technique systems
Make gold electrode lead and pin, (b) makes micro-pillar array with SU8 or PI, and (c) graphene transfer, (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 sites) of the present invention.
Fig. 4 is that mound shape graphene microelectrode site cyclic voltammetry scan curve of the present invention (have chosen three microelectrode positions
Point).
Fig. 5 is that present invention inverted microscope observes the people nerve mother being incubated on graphene three-dimensional micro-electrode array chip
Cell carcinoma cells (SH-SY5Y), and with multi channel signals detecting system detect cell spontaneous action potentials.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
Graphene three-dimensional micro-electrode chip fabrication technique flow is as shown in Fig. 2 specific as follows：
(1) substrate is cleaned：Using Phiranha solution cleaning silicon chip or quartz plate or Pyrex piece (such as Prex7740),
Rinsed well again with deionized water, nitrogen drying, oxygen plasma is handled 5 minutes；
(2) Lift-off techniques make outer microelectrode array region external electrode lead and pin：The spin coating on substrate
AZ4620, photoetching process is patterned, then splash-proofing sputtering metal layer (titanium/gold), is removed photoresist with acetone, is left metal figure
Layer.(Fig. 2 .a)
(3) three-dimensional micro-pillar array makes：Spin coating negative photoresist (silicon chip SU8 3005, quartz plate and Prex7740 boron
Silica glass PI 7510), control after rotating speed 3000r/min, photoetching development, by solidification, form micro-pillar array.(Fig. 2 .b)
(4) graphene is shifted：The single-layer graphene film prepared using chemical vapor deposition, be actually " copper/graphene/
Poly- methyl acid methyl esters " composite membrane；Laminated film ammonium persulfate immersion corrosion copper foil, after copper is corroded completely, leaves branch
Support layer " graphene/poly- methyl acid methyl esters " film；Film is rinsed with deionized water, then complete film is transferred to three-dimensional microtrabeculae
On array, cover array and with gold electrode wire contacts；Stand after a period of time, dried 30 minutes with 85 DEG C of baking ovens, then third
Immersion removes poly- methyl acid methyl esters in ketone solution；Finally substrate is cleaned successively with ethanol, deionized water.(Fig. 2 .c)
(5) graphene microelectrode is made：Spin coating AZ4620P photoresists, stone is made by photoetching and oxygen plasma etch
Black alkene figure, then removes photoresist with acetone, substrate is cleaned with ethanol, deionized water successively.(Fig. 2 .d)
(6) electrode dielectric layer is made：Spin coating SU8 3005, 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 are specific as follows：
(1) using electrochemical workstation Gamry reference 600, the electrification of microelectrode is carried out using three-electrode system
Learn and characterize.
(2) one plastic chamber is bonded in substrate with PDMS, microelectrode array region is within the cavity.Noted in cavity
Enter PBS solution.
(3) using graphene three-dimensional micro-electrode site as working electrode, Ag/AgCl is as reference electrode, and platinum filament is as right
Electrode, the PBS solution described in three electrode immersions (2).
(4) when carrying out electrochemical analysis, the sinusoidal ac signal that electrochemical workstation output peak value is 50mV, frequency
Scope is from 0.01HZ to 1MHz.
(5) when carrying out cyclic voltammetric analysis, scanning voltage scope -0.5V-0.5V, sweep speed 100mV/s.
Electrochemical impedance testing result is as shown in figure 3, in 1kHz, graphene three-dimensional micro-electrode structure measures less resistance
Anti- value is 300K Ω or so, disclosure satisfy that the impedance conditions of cell detection.For conventional planar microelectrode structure, by exhausted
Its electrode site can be less than the height of insulating barrier after edge layer encapsulation, and electrod-array will be sunk structure one by one.Through this
The three-dimensional mound shape bulge-structure provided is provided, the surface area of electrode site is not only increased, while also ensure that cell to be measured
Tissue with electrode site effectively contact, it is to avoid because fitted between cell and electrode not sternly caused by charge leakage, improve detect
Signal to noise ratio.
Cyclic voltammetry curve is tested as shown in figure 4, measure multiple Graphene electrodes, has obtained repeated preferable CV figures,
Confirming the mound shape graphene microelectrode site of the invention provided, each is respectively provided with stable charge transport capability.This implementation
Though example only have chosen three microelectrode sites, dozens of mound shape electrode site is all suitable for.
Embodiment 3
Cell culture and detection are carried out on graphene three-dimensional micro-electrode array chip
(1) surface treatment of graphene three-dimensional micro-electrode array：Before inoculating cell, graphene three-dimensional micro-electrode is 75%
Ethanol solution in immersion one hour after, dried under uviol lamp；Then by poly d-lysine (PDL) solution according to 2 μ g/
cm2Concentration be added dropwise on graphene three-dimensional micro-electrode array surface, ambient temperature overnight, then with 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 dissociations of cellar culture, centrifugation, piping and druming, with 1.0 × 107cells/mm3Density be inoculated in
On graphene three-dimensional micro-electrode array chip.
(3) SH-SY5Y cell inductions break up：Because ATRA can induce SH-SY5Y to be divided into nerve cell, so
Nutrient solution, which is used, contains 10nM ATRAs, 10% hyclone, 0.11g/L Sodium Pyruvates, 300mg/L glutamine
MEM/F12 culture mediums；Because of vitamin A acid photo-labile, lucifuge in incubation changes liquid daily.
(4) cell detection：After culture 5 days, graphene three-dimensional micro-electrode array chip is fixed on multichannel electrical signal inspection
In the fixture of examining system, then fixture is put on the sample stage of inverted microscope, record is thin while imaging is carried out
The spontaneous action potentials that born of the same parents produce, testing result is as shown in Figure 5.
Claims (4)
1. a kind of method for making graphene three-dimensional micro-electrode chip, it is characterised in that comprise the concrete steps that：
(1) substrate is cleaned：Using Phiranha solution cleaning silicon chip, quartz plate or Pyrex piece, then use deionized water rinsing
Totally, nitrogen is dried up, and oxygen plasma is handled 5 minutes；
(2) microelectrode array region external electrode lead and pin are made with Lift-off stripping technologies：The spin coating in substrate
AZ4620, photoetching process is patterned, then sputtered titanium/gold metal layer, is removed photoresist with acetone, is left metal figure layer；
(3) three-dimensional micro-pillar array makes：Spin coating negative photoresist, is controlled after rotating speed 2500-3500r/min, photoetching development, is passed through
Solidification, forms micro-pillar array；
(4) graphene is shifted：Single-layer graphene film is prepared using chemical vapor deposition, copper/graphene/poly- methyl acid first is obtained
Ester laminated film；Laminated film ammonium persulfate immersion corrosion copper foil, after copper is corroded completely, leaves supporting layer graphite
Alkene/poly- methyl acid methyl esters film；Film is rinsed with deionized water again, then complete film is transferred in three-dimensional micro-pillar array,
Cover array and with gold electrode wire contacts；Stand after a period of time, dried 30 minutes with 85 DEG C of baking ovens, then in acetone soln
Immersion removes poly- methyl acid methyl esters；Finally substrate is cleaned successively with ethanol, deionized water；
(5) graphene microelectrode is made：Spin coating AZ4620P photoresists, graphene is made by photoetching and oxygen plasma etch
Figure, then removes photoresist with acetone, substrate is cleaned with ethanol, deionized water successively；
(6) electrode dielectric layer is made：Spin coating SU8 3005, carries out photoetching, development, exposes graphene microelectrode site and gold electricity
Pole pin.
2. the method as described in claim 1, it is characterised in that：Pyrex piece described in above-mentioned steps (1) is Prex
7740。
3. the method as described in claim 1, it is characterised in that：In above-mentioned steps (3), when three-dimensional micro-pillar array makes, when
During using silicon chip as substrate, using the negative photoresists of SU8 3005, when using quartz plate or Pyrex piece as substrate, using PI
7510 negative photoresists.
4. the method as described in claim 1, it is characterised in that：Obtained graphene three-dimensional micro-electrode array on a transparent substrate
It is easy to be observed with inverted microscope.
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| CN106335234B (en) * | 2016-08-19 | 2018-10-19 | 中国科学院上海微系统与信息技术研究所 | A kind of graphene albumen laminated film and preparation method based on non-covalent modification |
| WO2018081657A2 (en) * | 2016-10-27 | 2018-05-03 | Koniku Inc. | Systems for detection |
| CN107219278B (en) * | 2017-05-19 | 2019-04-05 | 浙江理工大学 | A kind of detector and its detection method of cell releaser |
| CN107475072B (en) * | 2017-08-24 | 2020-12-11 | 浙江理工大学 | Cell dynamic characteristic monitoring system and method based on three-dimensional graphene interface electrode |
| CN109468220B (en) * | 2018-11-15 | 2020-05-01 | 江南大学 | Graphene chip for real-time detection of biological information of microbial cells |
| CN109655084B (en) * | 2018-12-10 | 2020-06-02 | 上海交通大学 | Preparation method of drivable nerve photoelectrode array |
| CN113061524B (en) * | 2020-06-17 | 2024-01-02 | 山东大学 | Double-layer microfluidic chip and breast cancer miRNA detection kit |
| CN114632559B (en) * | 2022-01-26 | 2023-03-31 | 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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Effective date of registration: 20230531 Address after: 201108 3rd floor, building 2 (b), 515 Shennan Road, Minhang District, Shanghai Patentee after: Shanghai prospective Innovation Research Institute Co.,Ltd. Address before: 200050 No. 865, Changning Road, Shanghai, Changning District Patentee before: SHANGHAI INSTITUTE OF MICROSYSTEM AND INFORMATION TECHNOLOGY, CHINESE ACADEMY OF SCIENCES |