CN107629958B

CN107629958B - Microfluidic chip with three-dimensional graphene interface and preparation method thereof

Info

Publication number: CN107629958B
Application number: CN201710670200.1A
Authority: CN
Inventors: 刘爱萍; 陈本永; 王夏华; 吴化平; 邢赟; 许为中; 周奇
Original assignee: Zhejiang Sci Tech University ZSTU
Current assignee: Tongxiang Beite Technology Co.,Ltd.
Priority date: 2017-08-08
Filing date: 2017-08-08
Publication date: 2020-07-14
Anticipated expiration: 2037-08-08
Also published as: CN107629958A

Abstract

The invention discloses a micro-fluidic chip with a three-dimensional graphene interface and a preparation method thereof, the micro-fluidic chip realizes the capture of single cells and double cells by designing micro-column capture grooves with different sizes and utilizing fluid dynamics, and impedance electric signals during micro-motion (such as cell adsorption, cell migration, cell proliferation and cell proliferation) of single cells or two cells are quantitatively acquired in real time by a three-dimensional graphene micro-column electrode, so that a new thought and method are provided for cancer pathology and single cell detection. The preparation of the microfluidic chip comprises the preparation and processing of links such as a patterned gold electrode array, a three-dimensional graphene micro-column electrode array, a PDMS flow channel, an openable PDMS cover plate and the like. The micro-fluidic chip processed by the method has good biocompatibility, the preparation method is mild in condition, simple and feasible, the process parameters are controllable, and the detected single cell electric signal intensity is 2 times that of the common micro-fluidic chip.

Description

Microfluidic chip with three-dimensional graphene interface and preparation method thereof

Technical Field

The invention belongs to the field of micro-nano processing (MEMS) and sensor preparation, and particularly relates to a micro-fluidic chip with a three-dimensional graphene interface and a preparation method thereof.

Background

Microfluidic chips refer to chemical or biological laboratories built on a chip of a few square centimeters. It integrates the basic operation units of sample preparation, reaction, separation, detection, cell culture, sorting, lysis, etc. in the fields of chemistry and biology, etc. into a very small chip for implementing various functions of conventional chemical or biological laboratories.

Microfluidic chips are characterized primarily by their active structure (channels, reaction chambers and other functional components) that contain fluids, which are on the order of microns in at least one dimension. Due to the micro-scale structure, the fluid exhibits and develops specific properties therein that differ from those of the macro-scale. Thus, a unique analysis can be used to generate a particular performance. The microfluidic chip has the characteristics of controllable liquid flow, extremely less consumed samples and reagents, ten-fold and hundred-fold improvement of analysis speed and the like, can simultaneously analyze hundreds of samples in several minutes or even shorter time, can realize the whole process of sample pretreatment and analysis on line, and the preparation method of the prior microfluidic chip mainly prepares a plurality of simple chips with nano-sized flow channels by methods such as photoetching, hot pressing and the like, and then bonds the chips with a cover plate for simple cell capture research. However, the current methods of photolithography, hot pressing, etc. can only obtain a planar two-dimensional structure, so that a real three-dimensional flow channel is difficult to obtain, and the three-dimensional micro flow channel can realize better reagent mixing and mutual reaction of different reagents. The use of a three-dimensional microchannel facilitates culturing, stimulation, observation, etc. of cells from various directions if a cell culture solution is introduced into the microchannel. Therefore, the three-dimensional micro-channel has more important application value. In recent years, micro-fluidic chips have received much attention from researchers, such as sensors based on micro-fluidic chips, centrifugal micro-fluidic chips for preparing droplets, paper micro-fluidic chips, and the like. Little attention has been paid to the study of microfluidic chips for cell sensing performance, especially single cells. Thus, the electrodes of the chip are an essential part of the chip, and the electrodes are involved in capturing cells and detecting and conducting cell signals. At present, most of sensing electrodes of microfluidic chips are of a planar two-dimensional gold electrode structure classically, the contact area of the electrodes and cells is small, the obtained cell signals are weak, and the micro-movement and migration of the cells in the vertical direction cannot be reflected, so that the study on the biological characteristics of the cells, particularly single cells, is not facilitated. And the stripping process and the photoetching technology are fully utilized. The microfluidic chip integrated with the three-dimensional graphene interface is manufactured by magnetron sputtering, reactive ion etching technology, particularly atomization deposition graphene film and other production processes, can be used for high-efficiency single cell capture and ultrasensitive single cell electric signal detection, and has certain guiding significance for research and diagnosis of cancer cells. And for single cell detection, the micro-fluidic chip eliminates the great influence of the traditional chip on the activity of cells by complex processes of chemical modification, chemical labeling and the like, and has complex operation and harsh conditions.

Disclosure of Invention

The invention aims to provide a micro-fluidic chip with a three-dimensional graphene interface and a preparation method thereof, aiming at the defects of the prior art. The micro-fluidic chip ensures high-efficiency single cell capture efficiency, obviously enhances the sensitivity of single cell sensing after capture, and improves the electrical signal sensing performance of single cells by one time compared with the traditional plane gold electrode.

The purpose of the invention is realized by the following technical scheme: a microfluidic chip with a three-dimensional graphene interface comprises a sub-chip, wherein the sub-chip is arranged in a test flow channel, and two ends of the test flow channel are respectively connected with a liquid storage tank; the two reservoirs and the test flow channel form an H-shaped flow channel. Cell electrodes are arranged on the sub-chip and are symmetrically arranged relative to a central reference electrode arranged at the position of the symmetry axis of the sub-chip, one side of the cell electrodes forms a working electrode, the other side of the cell electrodes forms a counter electrode, and the capture directions of the cell electrodes on the two sides are opposite and are perpendicular to the central reference electrode. The cell electrodes are mutually independent and are respectively connected with wiring terminals arranged at the edge of the microfluidic chip through leads; the cell electrode comprises an electrode base and a capture groove positioned on the electrode base, and the height of the capture groove is 30 μm; consists of a plurality of microelectrodes, and a gap of 5 μm is formed between adjacent microelectrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface which is vertical to the electrode base, and the arc is a semi-ellipse divided along a short axis. The electrode base comprises a gold layer and a graphene layer positioned on the upper surface of the gold layer, the arc-shaped capturing surface of the capturing structure is covered with the graphene layer, and the graphene layer is communicated with the gold layer; the graphene layer is provided with micro-nano folds and texture structures matched with the filamentous pseudo-feet on the cell surface. The cell electrode is a single cell electrode or a double cell electrode, and for the single cell electrode, the minor axis length of a semiellipse corresponding to the arc capture surface of the cell electrode is 16-20 μm, and the major axis length is 32-36 μm; for the double-cell electrode, the minor axis length of the semiellipse corresponding to the arc capture surface is 27-33 μm, and the major axis length is 40-45 μm.

A preparation method of a microfluidic chip of a three-dimensional graphene interface comprises the steps of constructing an H-shaped PDMS flow channel formed by two liquid storage tanks and a test flow channel positioned between the two liquid storage tanks, and fixedly arranging a sub-chip of cell electrodes in the test flow channel; the preparation method of the cell electrode comprises the following steps:

(1) forming a plurality of electrode units independent of each other on a glass substrate by a lift-off process; the electrode unit has a three-layer structure and sequentially comprises Ti/Au/Cr from top to bottom.

(2) And (4) washing the titanium layer by using hydrofluoric acid with the mass fraction of 1% to obtain the complete patterned gold electrode array.

(3) Forming a trap groove perpendicular to the electrode unit on the electrode unit by soft lithography, the trap groove being composed of a plurality of micro-electrodes with a gap of 5 μm between adjacent micro-electrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface which is vertical to the electrode unit, and the arc is a semi-ellipse divided along a short axis.

(4) With O₂Treating with 20W plasma for 1 min, and soaking in water10ml of a 20% aqueous solution of polydiallyldimethylammonium chloride (PDDA) was added for 20min and left to stand in an incubator at 35 ℃ for 1 hour.

(5) After washing and drying, 300 μ L1 mg/m L in GO solution was sprayed by atomization, dried at 40 ℃ for 12 hours at room temperature, and then reduced.

(6) Annealing was carried out at 200 ℃ for 2 hours in a dry nitrogen stream.

(7) Using 0.8J/cm²And patterning the continuous graphene film by using the laser beam with energy density to obtain the independent cell electrodes.

Further, the step 1 specifically comprises:

(1.1) spin coating a positive photoresist on the glass substrate at 2500rpm (S1818) to produce a PR layer 2 μm thick. The pattern on the reticle is transferred to a glass substrate by UV exposure and development.

(1.2) sequentially sputtering Cr/Au/Ti (10nm/100nm/100nm) on the glass substrate treated by the stripping process by a magnetron sputtering method.

(1.3) immersing in acetone solution for half an hour, then rinsing with ethanol and deionized water to completely wash off the photoresist layer to form a patterned gold electrode layer.

Further, in the step 5, hydrazine hydrate steam is used for reduction at 80 ℃ for 10 hours.

And further, the device also comprises a PDMS cover plate bonded to the PDMS flow channel, and the PDMS cover plate of the PDMS flow channel is processed by a template method.

Furthermore, the PDMS cover plate is divided into three parts by microdissection, and the part of the cover plate above the sub-chip can be opened for the microscopic recording of the cell morphology.

Further, the PDMS cover plate above the reservoir has openings, which are perforated by a hole puncher, for injecting or discharging the cell sap.

Further, the lead wires connecting the cell electrodes and the connection terminals were laid while constructing the electrode unit, and then SiO having a thickness of 600nm was deposited by Plasma Enhanced Chemical Vapor Deposition (PECVD)₂Coating, then using lightThe photoresist is used as a mask to block the region except the electrode unit, and Reactive Ion Etching (RIE) is used to etch away SiO on the electrode unit₂Layer, and lead wire is SiO₂And (4) covering with a coating.

The invention has the following advantages: the prepared three-dimensional reduced graphene oxide microcolumn electrode has an arc groove which is consistent with the size and the shape of a cell, the contact area of the cell and the electrode is remarkably increased, and compared with the traditional planar gold electrode, the contact between the cell and the electrode is extended to a three-dimensional space from a two-dimensional plane, so that the acquired electric signal can not only reflect the movement of the cell in the horizontal direction, but also reflect the migration of the cell in the vertical direction. Moreover, the nano structure and the texture of the graphene film interface are very consistent with the filamentous pseudo-podium microstructure on the cell surface, and the interaction between the cell and the electrode is increased, so that the sensing performance and the sensitivity are improved to a great extent, and the sensing performance is improved by one time on average compared with a sensing gold electrode; the design of the H-shaped PDMS flow channel can prevent the channel from being blocked when the cell buffer solution is injected. By opening the lid above the horizontal row of H-shaped channels, the object of the optical microscope can directly enter the internal flow channel, so that the movement of the cells can be observed very clearly. The micro-fluidic chip prepared by the invention can be recycled for multiple times, the utilization rate and the cost of the chip are improved, and the micro-fluidic chip has a good application prospect. The micro-fluidic chip processed by the method has good biocompatibility, the preparation method is mild in condition, simple and feasible, the process parameters are controllable, and the detected single cell electric signal intensity is 2 times that of the common micro-fluidic chip. The microfluidic chip prepared by the invention can be used for researching the characteristics of single cell such as adsorption, growth, proliferation and transfer by connecting an impedance analyzer and an optical microscope, and is mainly applied to the related fields of single cancer cell differentiation, drug resistance, cell morphology and the like in different canceration periods. Provides a new detection method for the cell detection field and provides a new research technology for cell biology.

Drawings

FIG. 1 is a schematic structural diagram of the whole microfluidic chip;

FIG. 2 is a plan view of a microfluidic chip;

FIG. 3 is an enlarged effect diagram of a capture area of a chip;

FIG. 4 is a schematic diagram of a single-cell impedance electrode;

fig. 5 is an effect diagram of capturing cells by a single-cell impedance electrode with a three-dimensional graphene bionic interface, and a graphene interface 4 with micro-nano folds and textures is clearly visible;

FIG. 6 is a process of formation of a patterned gold electrode array;

FIG. 7 is a process of processing different sizes of capture microcolumns by photolithography, followed by modifying the microcolumns with a graphene thin film and patterning it into individual sensing units by a laser beam;

FIG. 8 is a process of fabricating a PDMS runner and a PDMS cover plate using a standard template method, and bonding holes;

FIG. 9 is a real object diagram of the designed microfluidic chip and a microscopic image of the post-graphene-modified micro-column electrode;

fig. 10 is a performance characterization of the graphene microcolumn electrode and the entire microfluidic chip;

FIG. 11 is a photomicrograph of single and double cells captured on gold and graphene interface grooves;

FIG. 12 is a bode impedance spectrum and a phase spectrum of a single highly metastatic breast cancer cell (MDA-MB-231) at different stages of seeding on a 2D gold interface and a 3D graphene interface;

FIG. 13 shows the results of statistical analysis of the impedance amplitudes of five replicates (the mean. + -. variance of five replicates);

FIG. 14 is a simulated view of the flow velocity distribution inside and around the trapping microcolumn using finite element calculations;

FIG. 15 is a simulated plot of post-cell trapping electric field distribution using finite element calculations;

in the figure, a cell electrode 10, an electrode base 11, a capture chamber 12, a slit 13, a graphene layer 14, a central reference electrode 20, a test flow channel 21, a reservoir 22, and an opening 23 are shown.

Detailed Description

The invention aims to prepare a microfluidic chip with a three-dimensional graphene interface, which comprises an H-shaped PDMS flow channel formed by two liquid storage tanks and a test flow channel positioned between the two liquid storage tanks, and a sub-chip arranged in the test flow channel 21, as shown in FIG. 1, and can also comprise an openable PDMS cover plate (the shaded part in FIG. 1); the plane structure part is shown in fig. 2 and 3, the H-shaped PDMS channel can prevent the blocking of the microfluidic chip caused by the cell injection. The sub-chip includes a central reference electrode 20, and one or more mutually independent cell electrodes symmetrically disposed on both sides of the central reference electrode 20.

The sub-chip adopts a cell electrode 10 as shown in FIG. 4, which comprises an electrode base 11 and a capture groove 12 positioned on the electrode base 11, wherein the capture groove 12 is composed of a plurality of microelectrodes, and a gap 13 of 5 μm is arranged between adjacent microelectrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface which is vertical to the electrode base 11, and the arc is a semi-ellipse divided along a short axis. Wherein, the capture groove 12 is provided with a cambered groove which is matched with the size and the shape of the cell, and the contact point and the contact force of the cell membrane and the electrode material are increased to the maximum extent. Compared with the traditional plane gold electrode, the contact between the cell and the electrode is extended from a two-dimensional plane to a three-dimensional space, so that the collected electric signal can reflect the movement of the cell in the horizontal direction and the migration of the cell in the vertical direction. The design of the slit 13 allows the cell buffer to flow, but does not discharge the cells, so as to ensure that the arc-shaped capturing area of the micro-column does not accumulate too much cell buffer to affect the cell capturing efficiency.

As shown in fig. 5, the electrode base 11 includes a gold layer and a graphene layer 14 on the upper surface of the gold layer, and forms a three-dimensional graphene biomimetic interface. The graphene layer 14 has micro-nano folds and texture structures matched with the filamentous pseudo-feet on the cell surface, so that the topographic interaction and texture effect between the cells and the electrode material can be greatly enhanced, and the cell sensing performance is remarkably improved.

The preparation method comprises the following steps: (1) forming a plurality of mutually independent electrode units on the glass substrate through a stripping process for constructing an electrode base 11, wherein a plurality of leads for connecting the electrodes and the chip edge wiring terminals can be designed; the electrode has a three-layer structure and sequentially comprises Ti/Au/Cr from top to bottom. As shown in fig. 6, the following method is generally employed:

(1.1) spin coating positive photolithography (S1818) at 2500rpm on a glass substrate (fig. 6a) to produce a PR layer 2 μm thick (fig. 6b), transferring the pattern on the reticle onto the glass substrate by UV exposure (fig. 6c) and development (fig. 6 d).

(1.2) separately sputtering Cr/Au/Ti (10nm/100nm/100nm) on the glass substrate treated by the lift-off process by magnetron sputtering (FIG. 6 e). Where chromium serves as a bonding layer for glass and gold and titanium serves as a barrier layer for reactive ion etching.

(1.3) the sputtered substrate chip was immersed in an acetone solution for half an hour and then rinsed with ethanol and deionized water to completely wash away the underlying photoresist layer, forming a patterned gold electrode layer (fig. 6 f).

(2) To eliminate the interference of the leads with the signal of a single cell, a series of operations are required to cover all the leads, exposing only the area where the cells are captured. Deposition of SiO by Plasma Enhanced Chemical Vapor Deposition (PECVD)₂Coating, and etching SiO on the gold electrode by reactive ion etching using photoresist as mask₂And (4) coating. The following methods are generally employed:

(2.1) depositing SiO with a thickness of 600nm by a Plasma Enhanced Chemical Vapor Deposition (PECVD) process₂On the finished substrate (fig. 6 j).

(2.2) Next, the same lift-off method was used to block the area outside the micro-electrode array using photoresist as a mask. The barrier layer on the microelectrode array is etched away using Reactive Ion Etching (RIE) to expose the capture area (fig. 6g, 6h, 6 i).

(2.3) the titanium protective layer was rinsed with 1% hydrofluoric acid by mass fraction to obtain a complete patterned gold electrode array (fig. 6 k).

(3) Forming a capture groove perpendicular to the electrode base 11 on the gold electrode by soft lithography, the capture groove 12 being composed of a plurality of microelectrodes with a gap 13 of 5 μm between adjacent microelectrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface which is vertical to the electrode base 11, and the arc is a semi-ellipse divided along a short axis. The method specifically comprises the following steps: a 30 μm thick negative photoresist SU-8 was spin coated on a planar substrate of the patterned gold electrode array (fig. 7a) and then developed by UV exposure (fig. 7c, fig. 7d) to produce an array of hollow semi-cylindrical micro-pillars (fig. 7e) about 30 μm high for trapping cells.

(4) In order to better modify the graphene film on the microcolumns, it is necessary to make the microcolumns of the photoresist hydrophilic, and to use O for the glass substrate after the treatment₂The plasma (Ke You, China) was treated for 1 minute at 20W power (fig. 7 f). The microchip was then soaked in 10ml of 20% polydiallyldimethylammonium chloride (PDDA) (Sigma Aldrich) aqueous solution for 20min (fig. 7g) for enhancing the adsorption of GO (graphene oxide) to the microcolumn and left to stand in an incubator at 35 ℃ for 1 hour. The chip was washed with distilled water several times and dried in an incubator at 60 ℃ for 3 hours.

(5) A GO solution with the concentration of 300 mu L1 mg/m L is sprayed on a capture position of a microfluidic chip through an atomization method (figure 7h), and is dried at the room temperature of 40 ℃ for 12 hours to obtain a hollow semi-cylindrical micro-column array uniformly wrapped by multiple layers of GO sheets, and then the GO modified chip is treated under hydrazine hydrate (Sigma) steam at the temperature of 80 ℃ for 10 hours to reduce the GO film into an rGO (reduced graphene oxide) film (figure 7 i).

(6) The entire chip was annealed at 200 ℃ for 2 hours in a dry nitrogen gas flow to enhance the ohmic contact between the gold electrode and the graphene.

(7) Using 0.8J/cm²The continuous graphene film was patterned with a laser beam of energy density to obtain individual independent cell electrodes (fig. 7 j).

The cell electrodes 10 may be designed to be symmetrically arranged about a central reference electrode (20) (2cm by 2mm) at the location of the chip's axis of symmetry, one side constituting the working electrode and the other side constituting the counter electrode, the capture directions of the cell electrodes being opposite. The cell electrodes are symmetrically designed to form a working electrode and a counter electrode respectively, the counter electrode can remove the influence of the impedance of the cell buffer solution on the cell impedance, namely the difference of the working electrode impedance value and the counter electrode impedance value results in the cell impedance, and the working electrode and the counter electrode can be used interchangeably, so that the service life of the chip is prolonged. When the device is used, the working electrode, the counter electrode and the reference electrode are respectively connected with an impedance instrument, and the impedance instrument outputs an electric signal to the reference electrode so as to apply an electric field; and simultaneously acquiring cell signals collected by the working electrode and the counter electrode. And through the reverse arrangement of the cell electrodes on the two sides, the electrical signals obtained by the electrodes on the two sides are differentiated, and then the impedance signals caused by the physiological behaviors of the cells are obtained.

The H-shaped flow channel is made of PDMS and can be obtained by template processing. Can prevent the flow channel from being blocked when the cells are injected. The manufacturing process is shown in fig. 8, and specifically comprises the following steps: (a) forming a pattern with the height of 40 mu m on a glass substrate by using a stripping process, then casting liquid PDMS on the substrate, and curing and stripping to form a PDMS flow channel with the height of 40 mu m; (b) the PDMS cover plate was formed by the same method. Finally, the two parts are aligned and bonded, and the whole chip is packaged as shown in fig. 1, including a flow channel, an injection hole, a leaving hole and a cover. In particular, the cover can be opened at any time, the distance between the microscope objective and the cell surface is ensured to be less than 1mm, and a large-magnification picture is taken when the state of a single cell changes.

Fig. 9 is a physical diagram of the designed microfluidic chip. (a) Is a physical diagram of the chip; (b) capturing a photomicrograph of the location; (c) the height of the capture microcolumn obtained by a shape measuring microscope is about 30 mu m and is slightly larger than that of a single cell; (d) is a microscopic picture of the micro-column electrode after modifying the graphene; (e) is a magnified micrograph of the micro-column slit; (f) the large-magnification scanning electron microscope photo of the inner wall of the microcolumn can show obvious surface texture; (g) the roughness of the inner wall of the microcolumn is obtained by an atomic force microscope; (h) obtaining a single cell capturing unit after femtosecond laser etching; (i) is a single cell capture unit after cutting.

FIG. 10 is a representation of the performance of the micro-chamber including Raman, four probe resistance, voltammetry curves. (a) And (b) Raman testing of the positions of the plane and the microcolumn respectively, which shows that the graphene oxide is continuously modified on the plane and the microcolumn and is reduced; (c) the conductivity of the graphene oxide and the reduced graphene oxide films with different thicknesses is measured by a four-probe method, wherein the resistance of the reduced graphene oxide film with the thickness of 2 mu m reaches kilo ohms, and the signal of a single cell can be basically sensed; (d) the method is used for testing the cyclic voltammetry curves of the graphene oxide and the graphene film, the graphene oxide basically has no electrochemical activity, and the graphene film has an obvious redox peak, so that the prepared graphene microcolumn has good electrochemical activity.

Fig. 11 is a micrograph of single and double cell capture on gold and graphene interface grooves. (a) Capturing single cells by a gold interface; (b) gold interface capture of double cells; (c, d) capturing a plurality of single cells and double cells at the graphene interface respectively; after the cells are captured on a graphene interface, the single cells are tightly wrapped by the grooves of the graphene microcolumn (e) and obviously pressed and contacted with the inner wall of the microcolumn to generate a certain number of cell filamentous pseudo-podium (g); the double cells are tightly wrapped by the grooves of the graphene microcolumn (f) and are obviously in pressing contact with the inner wall of the microcolumn to generate a certain number of cell filopodia (h).

Fig. 12 shows different stages of seeding of single highly metastatic breast cancer cells (MDA-MB-231) on a 2D gold interface and a 3D graphene interface of the invention, including: bode impedance and phase profiles for loading, capture, attachment (culture for 2h), migration (culture for 5h), and proliferation (culture for 9 h). The graph shows that the impedance value and the phase value of the graphene interface are higher than those of the gold interface, more importantly, the distance between curves on the graphene interface is larger, and the fact that each physiological behavior of the cell reacts on the graphene interface more strongly is proved.

Fig. 13 is a graph of a single highly metastatic breast cancer cell (MDA-MB-231) seeded at different stages on a 2D gold interface and a 3D graphene interface of the invention, including: statistical analysis of impedance amplitudes for five replicates of no-load, capture, attachment (culture 2h), migration (culture 5h), and proliferation (culture 9h) resulted (shown as mean ± variance for five replicates). As can be seen from the figure, the sensing effect of the graphene interface is 2 times that of the classical gold interface.

The inner space of each arc-shaped groove can only contain one cell or two cells, and the speed difference between the capture region and the external slit, so the probability of capturing the cells by the designed capture structure can be proved to be 100%.

TABLE 1

Parameters	Values
		cell diameter	20μm
cell relative permittivity	200
		cell conductivity	0.84S/m
medium relative permittivity	80
		medium conductivity	0.01S/m
graphene relative permittivity	4.5
		graphene conductivity	6.5S/m

Claims

1. A microfluidic chip with a three-dimensional graphene interface comprises a sub-chip, wherein the sub-chip is arranged in a test flow channel (21), and two ends of the test flow channel (21) are respectively connected with a liquid storage tank (22); the two liquid storage tanks (22) and the test flow channel (11) form an H-shaped flow channel; cell electrodes are arranged on the sub-chip, the cell electrodes (10) are symmetrically arranged about a central reference electrode (20) arranged at the position of the symmetry axis of the sub-chip, one side of each cell electrode forms a working electrode, the other side of each cell electrode forms a counter electrode, and the cell electrodes (10) on the two sides have opposite capture directions and are perpendicular to the central reference electrode (20); the cell electrodes (10) are mutually independent and are respectively connected with wiring terminals arranged at the edge of the microfluidic chip through leads; the cell electrode (10) comprises an electrode base (11) and a capture groove (12) positioned on the electrode base (11), wherein the height of the capture groove (12) is 30 mu m; consists of a plurality of microelectrodes, with a gap (13) of 5 μm between adjacent microelectrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface which is vertical to the electrode base (11), and the arc is a semi-ellipse divided along a short axis; the electrode base (11) comprises a gold layer and a graphene layer (14) positioned on the upper surface of the gold layer, the graphene layer (14) covers the arc-shaped capturing surface of the capturing structure, and the graphene layer (14) is communicated with the gold layer; the graphene layer (14) is provided with micro-nano folds and texture structures matched with filamentous pseudo-feet on the cell surface; the cell electrode (10) is a single cell electrode or a double cell electrode, and for the single cell electrode, the minor axis length of a semiellipse corresponding to the arc capture surface is 16-20 μm, and the major axis length is 32-36 μm; for the double-cell electrode, the minor axis length of the semiellipse corresponding to the arc capture surface is 27-33 μm, and the major axis length is 40-45 μm.

2. The preparation method of the microfluidic chip with the three-dimensional graphene interface is characterized by comprising the steps of constructing an H-shaped PDMS flow channel consisting of two liquid storage tanks and a test flow channel (21) positioned between the two liquid storage tanks (22), and fixedly arranging a sub-chip with cell electrodes (10) in the test flow channel (21); the preparation method of the cell electrode (10) comprises the following steps:

(1) forming a plurality of electrode units independent of each other on a glass substrate by a lift-off process; the electrode unit has a three-layer structure and sequentially comprises Ti/Au/Cr from top to bottom;

(2) washing the titanium layer by using hydrofluoric acid with the mass fraction of 1% to obtain a complete patterned gold electrode array;

(3) forming a trap groove perpendicular to the electrode unit on the electrode unit by soft lithography, the trap groove (12) being composed of a plurality of micro-electrodes with a gap (13) of 5 μm between adjacent micro-electrodes; a plurality of microelectrodes are orderly arranged to form an arc capture surface vertical to the electrode unit, and the arc is a semi-ellipse divided along a short axis;

(4) with O₂Treating the plasma with 20W power for 1 minute, soaking in 10ml of 20% polydiallyldimethylammonium chloride (PDDA) water solution for 20min, and standing in an incubator at 35 ℃ for 1 hour;

(5) after washing and drying, spraying a GO solution with the concentration of 300 mu L1 mg/m L by an atomization method, drying at the room temperature of 40 ℃ for 12 hours, and then reducing GO;

(6) annealing in a dry nitrogen stream at 200 ℃ for 2 hours;

3. The method according to claim 2, wherein step 1 is specifically:

(1.1) spin-coating a positive photoresist S1818 on a glass substrate at 2500rpm to produce a PR layer 2 μm thick, transferring the pattern on the reticle onto the glass substrate by UV exposure and development;

(1.2) sequentially sputtering Cr/Au/Ti on the glass substrate treated by the stripping process by a magnetron sputtering method, wherein the thickness of the Cr/Au/Ti is 10nm/100nm/100 nm;

4. The method of claim 2, wherein in step 5, hydrazine hydrate steam is used for reduction at 80 ℃ for 10 hours.

5. The method of claim 2, further comprising a PDMS cover bonded to the PDMS channels, wherein the PDMS cover of the PDMS channels are processed by a template method.

6. The method of claim 5, wherein the PDMS cover is divided into three parts by microdissection, and the part of the cover above the daughter chip can be opened for microscopic recording of the cell morphology.

7. The method of claim 6, wherein the PDMS cover plate above the reservoir has openings, which are perforated by a hole puncher, for injecting or discharging the cell sap.

8. Method according to claim 2, characterized in that the leads connecting the cell electrodes (10) and the terminals are laid while constructing the electrode unit, and then SiO with a thickness of 600nm is deposited by Plasma Enhanced Chemical Vapor Deposition (PECVD)₂Coating, blocking the region outside the electrode unit using photoresist as a mask, and etching away SiO on the electrode unit using Reactive Ion Etching (RIE)₂Layer, and lead wire is SiO₂And (4) covering with a coating.