CN105460882A - Graphene three-dimensional microelectrode array chip, and method and application thereof - Google Patents

Abstract

The invention relates to a graphene three-dimensional microelectrode array chip, a method and an application thereof. It is characterized in that the described microcolumn array is made by using negative photoresist, and the microcolumn array is covered with a single-layer graphene film to produce a microelectrode array; the microelectrode chip includes a transparent graphene three-dimensional electrode array area and The peripheral gold electrode lead pins are in two parts. The microelectrode sites are three-dimensional protrusions. The three-dimensional microelectrode mound-shaped (or called hill-like) microelectrode structure is conducive to the formation of tight electrical coupling between rigid microelectrode sites and soft cells or tissues, and the excellent electrical properties of graphene can improve the microelectrode array. Electrophysiological detection sensitivity. In addition, the graphene three-dimensional microelectrode array fabricated on a transparent substrate is convenient for observation with an inverted microscope, which facilitates the application of various cell microscopic imaging methods and the use in combination with microfluidic chips.

Description

A graphene three-dimensional microelectrode array chip, method and application thereof

technical field

The invention relates to a graphene three-dimensional microelectrode array chip, a preparation method and an application thereof. The graphene three-dimensional microelectrode array chip provided by the invention can be used in the field of biomacromolecule, cell or tissue detection, especially in the detection of cells and tissues Electrophysiological testing.

Background technique

The microelectrode array chip is a sensor that integrates multiple micron-level electrodes on a chip for biological signal detection by using a micromachining process. The microelectrode array chip can simultaneously detect the electrical signals of biological macromolecules in the solution, detect the biological electrical signals of cells and tissues, or be used to electrically stimulate cells and tissues.

At present, the electrode materials of microelectrode array chips are mostly metals such as gold and platinum, but their opaque nature cannot be compatible with the inverted microscope widely used in cell biology. When using such a microelectrode array chip to detect the electrophysiological activity of cells or tissue slices, it is necessary to use an upright microscope equipped with a water immersion objective lens, which is inconvenient to operate and the tested samples cannot be further cultured and must be discarded. At present, based on good conductivity and transparency, tin-doped indium oxide (ITO) has become the most important transparent conductive material, which can be used to make micro-electrode array chips. However, compared with gold and platinum metal microelectrodes, ITO microelectrodes have disadvantages such as high electrochemical impedance and poor electrochemical stability in solution. The development of high-performance transparent microelectrode array chips is an urgent problem to be solved. The emerging hot material graphene has excellent properties such as high conductivity, high light transmittance, high mechanical strength, good electrochemical stability and good biocompatibility, and has begun to be used to make microelectrode arrays, and can detect Action potentials to neurons (XiaoweiDu, LeiWu, JiCheng, ShanluoHuang, QiCai, QinghuiJin, JianlongZhao, Graphenemicroelectrode arrays for neural activity detection, JBiolPhys (2015) 41:339–347). At present, the electrode sites of planar graphene microelectrode arrays cannot form close contact with soft cells and tissues, which affects the sensitivity of electrophysiological detection. Recent theoretical studies have shown that the morphology of single-layer graphene on the substrate is not affected by the hardness of the substrate and the change of the amplitude of the substrate, and it shows the morphological characteristics of the substrate surface (Liting Xiong, Yuanwen Gao. Surface roughness and size effect on the morphology of graphene substrate. PhysicaE:Low-dimensionalSystemSystemsandNanostructures. , 172(1-2):154-161). Based on this, this application intends to propose a chip, method and application for fabricating a three-dimensional graphene microelectrode array, in order to further improve the performance of the microelectrode array.

Contents of the invention

In order to overcome the shortcoming that the electrode sites of the existing graphene microelectrode array chip plane cannot form close contact with flexible cells or tissues, and increase the specific surface area of the electrode sites, the object of the present invention is to provide a graphene three-dimensional microelectrode array The chip, method and application thereof can be specifically applied to the detection of electrophysiological signals of cells and tissues.

The object of the present invention is achieved through the following measures: the chip comprises two parts (Fig. 1) of the transparent graphene three-dimensional microelectrode array area and the peripheral gold electrode lead pin area; the chip is made of silicon, quartz or borosilicate Glass is used as the substrate, and gold electrode leads and pins are fabricated by lift-off process outside the microelectrode array area (Fig. 2.a); SU-8 or PI micropillar arrays are fabricated in the microelectrode array area (Fig. 2.b, column The ratio of height to diameter is about 0.3), and then the graphene/polymethyl methate film is transferred to the microelectrode array area to form a good ohmic contact with the prefabricated gold electrode leads on the substrate (Fig. 2.c), Then use AZ4620 photoresist as the mask layer for graphene patterning, and use oxygen plasma (OxygenPlasma) to etch the graphene (Fig. 2.d). Electrode sites and two-dimensional electrode leads; finally, use PI photoresist to make an insulating layer on the surface of the patterned graphene and the surface of the gold electrode leads, exposing the three-dimensional mound-shaped graphene micro-electrode sites and gold electrode leads (Fig. 2.e).

The present invention has the advantages that several to dozens of graphene three-dimensional microelectrode array sites can be produced at the same time; the photoresist microcolumn has smooth edges, which can lift up the graphene film without damaging the graphene film; cells and tissues It can wrap the graphene microelectrode sites in the shape of hills (or called hills, the same below), and improve the ability of microelectrodes to detect weak electrical signals; use graphene's excellent electrical properties, high light transmittance, and excellent electrochemical properties The transparent microelectrode array chip made of stability and biocompatibility is convenient to be used in combination with an inverted microscope, so that many biological imaging methods such as high-resolution fluorescence imaging can be combined with electrophysiological detection.

In summary, the present invention relates to a graphene three-dimensional microelectrode array chip, method and application thereof. It is characterized in that the described microcolumn array is made by using negative photoresist, and the microcolumn array is covered with a single-layer graphene film to produce a microelectrode array; the microelectrode chip includes a transparent graphene three-dimensional electrode array area and The peripheral gold electrode lead pins are in two parts. The microelectrode sites are three-dimensional protrusions. The three-dimensional microelectrode mound-shaped microelectrode structure is conducive to the tight electrical coupling between rigid microelectrode sites and soft cells or tissues, and the excellent electrical properties of graphene can improve the electrophysiological detection sensitivity of microelectrode arrays. In addition, the graphene three-dimensional microelectrode array fabricated on a transparent substrate is convenient for observation with an inverted microscope, which facilitates the application of various cell microscopic imaging methods and the use in combination with microfluidic chips.

Description of drawings

Figure 1 is a schematic diagram of a graphene three-dimensional microelectrode array chip of the present invention, a) is a physical map of the chip, and b) is a graphene three-dimensional microelectrode array area.

Fig. 2 is the manufacturing process flow chart of graphene three-dimensional microelectrode array chip of the present invention, (a) make gold electrode lead wire and pin with lift-off process, (b) make microcolumn array with SU8 or PI, (c) graphene transfer, (d) graphene patterning, (e) SU8 or PI to make insulating layer.

Fig. 3 is the electrochemical impedance spectrum of the mound-shaped graphene microelectrode site of the present invention (three microelectrode sites are selected).

Fig. 4 is the cyclic voltammetry scanning curve of the mound-shaped graphene microelectrode site of the present invention (three microelectrode sites are selected).

Figure 5 shows the observation of human neuroblastoma cells (SH-SY5Y) cultured on the graphene three-dimensional microelectrode array chip with an inverted microscope, and the spontaneous action potential of the cells is detected with a multi-channel signal detection system.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

The manufacturing process of graphene three-dimensional microelectrode chip is shown in Figure 2, and the details are as follows:

(1) Clean the substrate: use Phiranha solution to clean the silicon wafer or quartz wafer or borosilicate glass wafer (such as Prex7740), then rinse with deionized water, blow dry with nitrogen, and treat with oxygen plasma for 5 minutes;

(2) Lift-off process to make the outer electrode leads and pins in the outer microelectrode array area: Spin-coat AZ4620 on the substrate, pattern it by photolithography, then sputter the metal layer (titanium/gold), and remove the photolithography with acetone Glue, leaving the metal layer. (Figure 2.a)

(3) Production of three-dimensional microcolumn array: Spin-coat negative photoresist (SU83005 for silicon wafers, PI7510 for quartz wafers and Prex7740 borosilicate glass), control the rotation speed at 300r/min, and after photolithography and development, cure to form microcolumns array. (Figure 2.b)

(4) Graphene transfer: the single-layer graphene film prepared by chemical vapor deposition is actually a "copper/graphene/polymethyl methate" composite film; the composite film is soaked and corroded copper foil with ammonium persulfate, and the After the copper is completely etched, the support layer "graphene/polymethylmethacrylate" film is left; the film is rinsed with deionized water, and the complete film is transferred to the three-dimensional micropillar array, covering the array and connecting with the gold electrode lead Contact; after standing for a period of time, bake in an oven at 85°C for 30 minutes, then soak in acetone solution to remove polymethyl methate; finally wash the substrate with ethanol and deionized water in sequence. (Figure 2.c)

(5) Fabrication of graphene microelectrodes: spin-coat AZ4620P photoresist, make graphene patterns by photolithography and oxygen plasma etching, then remove the photoresist with acetone, and clean the substrate with ethanol and deionized water in turn. (Figure 2.d)

(6) Fabrication of the electrode insulation layer: spin-coat SU83005, perform photolithography and development, and expose the graphene micro-electrode sites and gold electrode pins. (Figure 2.e)

Example 2

Graphene three-dimensional microelectrode performance testing method

The electrochemical impedance and cyclic voltammetry curve test methods are as follows:

(1) The electrochemical workstation Gamryreference600 was used to perform electrochemical characterization of the microelectrodes using a three-electrode system.

(2) A plastic cavity is glued on the substrate with PDMS, and the microelectrode array area is located in the cavity. Inject PBS solution into the cavity.

(3) The graphene three-dimensional microelectrode site is used as the working electrode, the Ag/AgCl is used as the reference electrode, and the platinum wire is used as the counter electrode, and the three electrodes are immersed in the PBS solution described in (2).

(4) When performing electrochemical impedance analysis, the electrochemical workstation outputs a sinusoidal AC signal with a peak value of 50mV, and the frequency range is from 0.01HZ to 1MHz.

(5) When performing cyclic voltammetry analysis, the scanning voltage range is -0.5V-0.5V, and the scanning speed is 100mV/s.

The electrochemical impedance detection results are shown in Figure 3. At 1kHz, the graphene three-dimensional microelectrode structure measured a relatively small impedance value of about 300KΩ, which can meet the impedance conditions for cell detection. For the traditional planar microelectrode structure, the electrode sites will be lower than the height of the insulating layer after being encapsulated by the insulating layer, and the electrode array will be a recessed structure one by one. The three-dimensional mound-shaped convex structure provided by the present invention not only increases the surface area of the electrode site, but also ensures the effective contact between the cell tissue to be tested and the electrode site, avoiding the damage caused by the loose fit between the cell and the electrode. The charge leakage can improve the signal-to-noise ratio of the detection.

The cyclic voltammetry curve test is shown in Figure 4, and a plurality of graphene electrodes are measured, and a CV figure with good repeatability is obtained, which confirms that each of the mound-shaped graphene microelectrode sites provided by the present invention has stable charge transfer ability. Although only three microelectrode sites are selected in this embodiment, it is applicable to dozens of mound-shaped electrode sites.

Example 3

Cell culture and detection on a graphene three-dimensional microelectrode array chip

(1) Surface treatment of the graphene three-dimensional microelectrode array: before seeding the cells, the graphene three-dimensional microelectrode was soaked in 75% ethanol solution for one hour, and dried under an ultraviolet lamp; then the poly-D-lysine (PDL) solution was added dropwise on the surface of the graphene three-dimensional microelectrode array at a concentration of 2 μg/cm ² , left overnight at room temperature, rinsed three times with ultrapure water, and air-dried.

(2) Seeding cells on the surface of graphene three-dimensional microelectrode array: Digest conventionally cultured SH-SY5Y cells with digestion solution (0.25% trypsin, 0.02% EDTA solution), centrifuge, and pipette at 1.0×10 ⁷ cells/mm ³ The density is seeded on the graphene three-dimensional microelectrode array chip.

(3) SH-SY5Y cell differentiation: because all-trans retinoic acid can induce SH-SY5Y to differentiate into nerve cells, so the culture medium adopts 10nM all-trans retinoic acid, 10% fetal bovine serum, 0.11g/L acetone MEM/F12 medium with sodium nitrate and 300mg/L glutamine; because retinoic acid is unstable in light, the medium should be protected from light during the cultivation process, and the medium should be changed every day.

(4) Cell detection: After 5 days of culture, fix the graphene three-dimensional microelectrode array chip in the fixture of the multi-channel electrical signal detection system, then place the fixture on the sample stage of the inverted microscope, and record the cell while performing imaging observation. The generated spontaneous action potential, the detection result is 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.