CN103901089B - The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method - Google Patents
The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method Download PDFInfo
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Abstract
The present invention relates to a kind of sensor detecting neurocyte electricity physiological signal and manufacture method and detection method.Described sensor includes substrate, cultivates cavity wall, graphene film, source electrode, drain electrode and reference electrode, described substrate and culture chamber wall seamless sealing-in surround culture chamber, it is attached with graphene film on substrate in described culture chamber, described graphene film can make neurocyte grow thereon through chemical modification, described source electrode and drain electrode electrically connect with described graphene film respectively and fix with described substrate respectively and be connected, and described reference electrode is placed in described culture chamber.Utilize sensor of the invention detection neurocyte electricity physiological signal that signal to noise ratio and spatial resolution can be greatly improved.
Description
Technical field
The present invention relates to bio-instruments field of engineering technology, in particular it relates to a kind of detection neurocyte electricity is raw
The sensor of reason signal and the manufacture method of described sensor, and use described sensor to detect neurocyte
The method of electricity physiological signal.
Background technology
Field-effect transistor be a kind of by field effect control electric current electronic component, have grid (gate),
Source electrode (source) and drain electrode (drain) three terminals, by putting on electric field produced by the voltage of grid
Control to connect the size of current circulated in the raceway groove of source electrode and drain electrode.The raceway groove of conventional field effect transistor by
Heavily doped silicon is constituted.
Along with the development of nanosecond science and technology, various novel low-dimension nano materials, such as silicon nanowires (SiNW),
CNT (CNT) and Graphene (graphene) etc., with the character of its uniqueness (such as skin effect, body
Long-pending effect and quantum size effect etc.) cause the extensive concern of people.Compared with traditional material, nanometer material
Material has less size, higher specific surface area, more excellent electrical properties and more preferable biocompatibility
Deng.When the raceway groove of conventional field effect transistor is substituted by nano material, i.e. constitute nano material field effect
Transistor, such as silicon nanowires field-effect transistor (SiNW-field-effect transistor), carbon nanotube field-effect
Transistor (CNT-field-effect transistor) and graphene field effect transistor (Gra-field-effect transistor) etc..
Graphene is a kind of emerging carbon nanomaterial, has that electrical conductivity is high, mechanical strength is big and electrochemistry is steady
The fixed physical and chemical performance waiting excellence so that it is there is in high-sensitivity detection field the application advantage of uniqueness, draw
Play people to pay close attention to greatly.Graphene is the planar crystal of monolayer atomic building, and each atom exists
On surface, the change of external environment all will directly affect all carbon atoms constituting Graphene so that it is to interface
Response the sensitiveest, simultaneously unique structure makes it have outstanding detection sensitivity.Develop at present
Highly sensitive nitric oxide (NO) gas detecting chip based on Graphene, has the high spy of single NO molecule
Survey sensitivity, show that Graphene has huge potentiality as detection chip sensing element.
The analysis of neuroelectricity signal is one of main contents of current nuroinformatics research.The most often
Device be microelectrode array sensor (Micro-Electrode Array, MEA).MEA is by being embedded in base
Microelectrode composition in substrate matter.Microelectrode mainly by metal material, such as platinum, gold, titanium-nitrogen-oxygen compound and
Indium tin oxides etc. form.Microelectrode is arranged in array on substrate.
Although can detect, with microelectrode array sensor, the electrical signal that neurocyte is provided well, but
The noise level obtained is inversely proportional to the size of microelectrode size, when microelectrode is reduced in size to certain size
Waiting, the level of its noise will substantially exceed the size of its signal, thus real signal be can not be identified.Therefore
The spatial resolution detecting neurocyte by the method is the highest.
Summary of the invention
For improving spatial resolution and the signal to noise ratio of detection neurocyte further, the present invention provides a kind of detection
The sensor of neurocyte electricity physiological signal and the manufacture method of described sensor, and use described sensor
The method of detection neurocyte electricity physiological signal.
Graphene is made the Graphene sensing element of graphene field effect transistor by the present inventor, and one
Aspect utilizes the high sensitivity of Graphene to improve the size of signal, and still further aspect field-effect transistor is not
Being limited to contact resistance, it is less that detector unit can be done, and sensor of the invention therefore can be utilized to enter one
Step improves signal to noise ratio and the spatial resolution of detection neurocyte.
For realizing the purpose of the present invention, the present invention provides technical scheme:
In first aspect, the present invention provides a kind of sensor detecting neurocyte electricity physiological signal, including base
Plate, cultivation cavity wall, graphene film, source electrode, drain electrode and reference electrode, described substrate and cultivation cavity wall
Seamless sealing-in surrounds culture chamber, and the substrate in described culture chamber is attached with graphene film, described graphene film
Neurocyte can be made to grow thereon through chemical modification, described source electrode and drain electrode respectively with described Graphene
Film electrically connects and fixes with described substrate respectively and is connected, and described reference electrode is placed in described culture chamber.
As the preferred version of the present invention, described chemical modification is poly-D-lysine and/or laminin，LN modification.
Preferably, described chemical modification is the non-covalent bond modification of poly-D-lysine and/or laminin，LN.
As the preferred version of the present invention, described substrate includes silicon chip basic unit and silicon dioxide surface layer, described stone
Ink alkene film is attached on described silicon dioxide surface layer.
As the preferred version of the present invention, the Graphene number of plies of described graphene film is one to two layer.
Preferably, the area of described graphene film is 1-200 μm2, such as 2 μm2、5μm2、8μm2、10μm2、
15μm2、20μm2、30μm2、50μm2、80μm2、100μm2、120μm2、140μm2、150μm2、
175μm2、185μm2、190μm2、195μm2, preferably 10-100 μm2, more preferably 20-30 μm2。
As the preferred version of the present invention, the distance between described source electrode and drain electrode is 500-5000nm,
600nm、800nm、1000nm、1200nm、1500nm、2000nm、2500nm、2800nm、3200nm、
3500nm, 4000nm, 4200nm, 4500nm, 4800nm, preferably 750-4000nm, more preferably
1000-3000nm。
As the preferred version of the present invention, described source electrode and drain electrode are each independently metal electrode.
Preferably, the material of described metal electrode is platinum, gold and/or chromium, can be a kind of metal material, also
It can be the alloy of two or more material.
Preferably, the thickness of described metal electrode is 0.03-0.15 μm, such as 0.04 μm, 0.05 μm, 0.06 μm,
0.08μm、0.10μm、0.11μm、0.12μm、0.13μm、0.14μm、0.15μm。
Preferably, described reference electrode is Ag/AgCl electrode.
As the preferred version of the present invention, the material of described cultivation cavity wall is polydimethylsiloxane (PDMS).
In second aspect, the present invention provides the detection neurocyte electricity physiological signal described in a kind of first aspect
The manufacture method of sensor, comprises the following steps:
(1) on substrate, graphene film is adhered to；
(2) on the substrate be attached to graphene film, source electrode and drain electrode, described source electrode and electric leakage are formed
Pole electrically connects with described graphene film respectively and fixes with described substrate respectively and is connected；
(3) by described be attached to graphene film and define the substrate of source electrode and drain electrode with cultivate cavity wall without
Seam to connect and surround culture chamber so that described graphene film, source electrode and drain electrode are respectively positioned in described culture chamber；
(4) described graphene film is chemically modified, so that neurocyte grows thereon.
In the third aspect, the present invention provides a kind of method detecting neurocyte electricity physiological signal, including following
Step:
(1) train in the culture chamber of the sensor of detection neurocyte electricity physiological signal as described in relation to the first aspect
Support neurocyte, make neurocyte be in close contact with graphene film；
(2) between described source electrode and drain electrode, constant channel voltage is applied, when the film electricity of neurocyte
When position changes, the electric current flowing through described graphene film produces change, by detecting on described graphene film
The transmembrane potential change of neurocyte is measured in the change of electric current.
As the preferred version of the present invention, the channel voltage applied between described source electrode and drain electrode is
10-70mV, such as 12mV, 15mV, 18mV, 22mV, 25mV, 30mV, 40mV, 50mV,
55mV, 58mV, 60mV, 65mV, 68mV, preferably 30-60mV.
The invention have the benefit that the present invention utilizes Graphene to make the graphite of graphene field effect transistor
Alkene sensing element, owing to Graphene has high sensitivity, utilizes sensor of the invention detection neurocyte electricity
Signal to noise ratio and the spatial resolution of physiological signal are greatly improved.It is experimentally confirmed that utilize sensor of the invention to examine
The signal to noise ratio of survey neurocyte electricity physiological signal is more than 5 times, and microelectrode array compared to existing technology senses
Device improves a lot.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and constitutes the part of description, with under
The detailed description of the invention in face is used for explaining the present invention together, but is not intended that limitation of the present invention.In accompanying drawing:
Fig. 1 is the process chart that the present invention adheres to graphene film on substrate.
Fig. 2 is the process chart that the present invention forms source electrode and drain electrode on substrate.
Fig. 3 is the cross-sectional of the substrate being attached to graphene film of the present invention.
Fig. 4 is the cross-sectional of the substrate sheltered with photoresist of the present invention.
Fig. 5 is the exposure of the present invention the cross-sectional of substrate after developing, it is shown that form electrode
Region.
Fig. 6 is the cross-sectional of the substrate defining source electrode and drain electrode of the present invention.
Fig. 7 is that the cross section defining source electrode and drain electrode the substrate that eliminates photoresist of the present invention shows
It is intended to.
Fig. 8 is being provided with culture chamber and being filled with the cross section of sensor of liquid in culture chamber of the present invention
Schematic diagram.
Fig. 9 is the schematic top plan view of sensor of the invention, does not include reference electrode.
Figure 10 is the optical microscope photograph of the sensing element of the present invention.
Figure 11 is the cross-sectional of sensor of the invention, including reference electrode Ag/AgCl.
Figure 12 is that the electric current in the sensing element of the present invention is along with the variation diagram of reference electrode voltage Vgate.
Figure 13 is the pass that the electric current in the sensing element of the present invention changes along with the pH value difference of liquid sample
Tying fruit figure.
Figure 14 is that the sensing element of the present invention forms, with the cell membrane of measured neurocyte, the signal being in close contact
Figure.
Figure 15 is the knot of the hippocampus neurocyte action potential of the mice arrived by the sensing element senses of the present invention
Fruit figure.
Description of reference numerals:
1-graphene film
The surface layer of 2-substrate
The basic unit of 3-substrate
4-photoresist
5-forms the region of source electrode
6-forms the region of drain electrode
7-source electrode
8-drain electrode
9-cultivates cavity wall
Detailed description of the invention
Below the detailed description of the invention of the present invention is described in detail.It should be appreciated that it is described herein
Detailed description of the invention be merely to illustrate and explain the present invention, be not limited to the present invention.
Sensor of the invention includes chip unit and cultivates cavity wall.Wherein, chip unit includes sensing element
And reference electrode.Described sensing element includes substrate and is fixed on the source electrode on described substrate and electric leakage
Graphene film it is electrically connected with between pole, and source electrode and drain electrode.
Wherein, described sensing element can be as the element of field effect transistor chemical biosensor.
Under precedence case, described sensing element method as follows prepares: (1) is adhered on substrate
Graphene film；(2) on the substrate be attached to graphene film formed electrode, described electrode include source electrode and
Drain electrode, and between source electrode and graphene film and between drain electrode and graphene film, form contact electricity
Connect.
Wherein, described graphene film can be conventional use of graphene film, the side being such as by mechanically pulling off
The graphene film that method obtains, it is also possible to the graphene film of chemical vapour deposition technique growth.
Wherein, the operation adhering to graphene film on substrate can be by pressing graphene film on substrate
Mode realizes.
Wherein it is possible under an optical microscope the graphene film on substrate is positioned also based on interference effect
Its border of labelling, it is also possible to utilize Raman spectrum and atomic force microscope that its number of plies is identified further.
The method according to the invention, wherein, under preferable case, in step (1), substrate includes basic unit and attached
The surface layer in basic unit, and graphene film is attached on the surface layer of substrate；Basic unit is silicon chip, and surface layer is
Silicon dioxide layer.Under this preferable case, substrate can by directly by preparing in the way of silicon chip is aoxidized,
It is also easy to carry out under an optical microscope the graphene film on substrate being carried out location labelling based on interference effect
Its border.
The method according to the invention, wherein, under precedence case, in step (2), specifically on substrate
The region forming source electrode forms source electrode, and the region of described formation source electrode includes being covered by graphene film
Region and the region not covered by graphene film；The region forming drain electrode on substrate forms drain electrode, institute
The region stating formation drain electrode includes the region covered by graphene film and the region not covered by graphene film；
Distance between described source electrode and drain electrode is 500-5000nm, preferably 750-4000nm, more preferably
For 1000-3000nm.Wherein, the distance between source electrode and drain electrode refers to adjacent source electrode and drain electrode
Between distance between immediate 2.
Wherein, under preferable case, in the region forming source electrode on substrate or the region forming drain electrode,
The area ratio in the region covered by graphene film and the region not covered by graphene film is 0.2-3:1, is preferably
0.5-1.5:1, more preferably 0.8-1.2:1.
Wherein, on substrate, the region covered by graphene film can be determined by observation by light microscope
Its border also positions.Boundary in the region that graphene film covers can select to include being covered by graphene film
Region and the region that do not covered by graphene film as forming the region of source electrode on substrate or forming electric leakage
The region of pole.After being selected on substrate the region of the source that formed (or leakage) electrode, can be by conventional electronics
The mode of bundle exposure exposes and forms the substrate regions of electrode and shelter the substrate regions that is formed without electrode, then leads to
Overheated evaporation or electron beam evaporation plating form source (or leakage) electrode.Can also be with organic molten after forming electrode
Shelter is removed in agent.Wherein, source electrode and drain electrode can be normal in field effect chemical biosensor field
The electrode that rule use, such as, can be to be deposited with by heat or metal electrode that electron beam evaporation plating obtains, and metal is electric
The material of pole can be at least one in platinum, gold and chromium, and the thickness of electrode can be 0.03 μm-0.15 μm.
Source electrode and drain electrode respectively and are provided with between substrate for fixing connection.Such as it is deposited with by heat or electricity
The metal electrode that son bundle evaporation obtains, includes, owing to forming the region of electrode, the region covered by graphene film
The region not covered by graphene film, the region on the one hand covered by graphene film defines electrode and graphite
Electrical connection between alkene film, the region on the other hand not covered by graphene film defines between electrode and substrate
For fixing connection.
The method according to the invention, wherein realizes seamless sealing-in between chip unit and cultivation cavity wall.A kind of excellent
The embodiment of choosing may is that first by organic with SYLGARD184 for polydimethylsiloxane (PDMS)
Silicon body, according to the ratio mix homogeneously of mass ratio 10:1, pours 10cm into2Culture dish in so that PDMS
With the upper surface flush of culture dish, then solidify 4-5 hours at 60 DEG C；Then the PDMS being cured
From culture dish take out, cut therebetween a length of side be 1.5 × 1.5cm, the degree of depth be the culture chamber of 1cm.Connect
And culture chamber oxygen plasma is processed, processing parameter is power 10-200W, and oxygen flow is
10-150sccm, the time is 10-120s.Then it is fixed on substrate after the culture chamber after process, it is ensured that pass
Sensor cell (source electrode, drain electrode and graphene film) is in culture chamber, and surrounding carries out nothing with PDMS again
Seam and connect.
The method according to the invention, removes residual harmful substance the sensor chip deionized water processed,
Then sensor chip being immersed in the ethanol that concentration is 75% and carry out sterilizing, the time is 2-10h, is preferably
3h。
The method according to the invention, grows on graphene film to realize neurocyte, needs Graphene
Film is modified accordingly.Typical case but the example of the modification of indefiniteness be such as: glues with poly-D-lysine or layer
Even at least one in albumen carries out non-covalent bond modification to graphene membrane surface, and wherein, poly-D-lysine is repaiied
The technological parameter of decorations is: poly-D-lysine concentration is 1mg/ml-20mg/ml, and the response time is 1-2 hour,
Modifying temperature is 37 DEG C, and pH value is 7.4；The modification parameter of laminin，LN is: laminin，LN concentration is
1mg/ml-10mg/ml, the modification time is 1-2 hour, and modifying temperature is 37 DEG C, and pH value is 7.4.Modify
After technique, use phosphate (PBS) wash buffer of sterilizing.
The method of the detection neurocyte electricity physiological signal that the present invention provides, comprises the following steps:
(1) in the culture chamber of the sensor of detection neurocyte electricity physiological signal of the present invention, god is cultivated
Through cell, neurocyte is made to be in close contact with graphene film；
(2) between described source electrode and drain electrode, constant channel voltage is applied, when the film electricity of neurocyte
When position changes, the electric current flowing through described graphene film produces change, by detecting on described graphene film
The transmembrane potential change of neurocyte is measured in the change of electric current.
The method according to the invention, wherein, separates and cultivates the neurocyte of hippocampus.It is referred to document
Stefanie K,Gary B.Culturing hippocampal neurons.Nature protocol,2006,1,
Method in 2406-2415 is carried out.Separating and cultivate other neurocyte can be according to technology well known in the art
Means are carried out.Described animal can be conventional laboratory animal, such as Amphibian, birds or mammal,
Include but not limited to Bufo siccus, chicken, mice, rat, dog, rabbit and monkey.
The method according to the invention, wherein, the voltage between source electrode and drain electrode can be 10-70mV,
It is preferably 30-60mV.
To be described the present invention by embodiment below.In following example, scanning electron microscopy
Mirror is purchased from HIT's model S-4800, and the tester of electrical properties is purchased from AXON company type of the U.S.
Number Axopatch200B electrical testing system, agents useful for same is the analytical reagent being purchased.
Preparation embodiment 1
With reference to Fig. 1-11, the present embodiment is used for illustrating to prepare in the method according to the invention the step of sensing element,
I.e. prepare the step of deflocculated graphite alkene fieldtron.
(1) with reference to Fig. 1, according to document Li X S, Cai W W, An J H, et al.Large-area synthesis
of high-quality and uniform graphene films on copper foils.Science,2009,324:
Method in 1312 1314 by the method for chemical gaseous phase deposition at the bottom of cuprio, (size is 4cm × 8cm, thick
Degree is 100 μm) on grow Graphene.Photoresist thick for one layer of 300nm of spin coating on the copper sheet have Graphene
(PMMA950K, purchased from ALLRESIST company of Germany), at 120 DEG C, baking makes solvent volatilize completely；
The copper sheet that spin coating has photoresist puts into copper etching liquid (containing 1mol/L ferric chloride and the solution of 1mol/L hydrochloric acid)
In be etched to copper reaction completely；Obtain the graphene film adhered on a photoresist.
(2) with reference to Fig. 1-3, by have 280nm thickness thermal oxide SiO2 silicon chip (size is 3cm × 3cm,
Thickness is 0.52cm, purchased from U.S. sillicon Valley Microelectrics) as substrate, i.e. basic unit 3 is
Silicon chip, surface layer 2 is the substrate of silicon dioxide layer.The graphene film that will be attached on photoresist transfers to substrate
On, and press make graphene film contact with substrates into intimate；Then by photoresist lysate (acetone) by stone
The photoresist on ink alkene film surface dissolves, and reservation graphene film, on substrate, obtains being attached with graphene film 1
Substrate (Fig. 3).Positioned under an optical microscope based on interference effect and mark boundaries, for really
It is shaped as the region of electrode.
(3) with reference to Fig. 4 and Fig. 5, the substrate being attached with graphene film 1 forms source electrode and drain electrode.
Specifically, the thick photoresist (PMMA of one layer of 300nm is revolved at the substrate surface being attached with graphene film 1
950K, purchased from ALLRESIST company of Germany), it is baked to solvent volatilization at 180 DEG C completely.According to optics
The border of the graphene film 1 of labelling under microscope, determines and forms the region 5 of source electrode and form drain electrode
Region 6, is formed in the region of source (or leakage) the electrode strip electrode of 2 μm (width be) by graphene film 1
The area ratio in the region covered and the region not covered by graphene film 1 is 1:1, with electron beam exposure apparatus to shape
The region of Cheng Yuan (or leakage) electrode is exposed, and at developer solution, (1:5 pressed by 4-methyl-2 pentanone and isopropanol
Volume ratio mixing mixed liquor) in development 10min, in isopropanol after fixing 10min, dry up with nitrogen；
Thus expose and form the substrate regions of electrode and shelter the substrate regions that is formed without electrode.
(4) with reference to Fig. 6, in high vacuum conditions, according to document (" film preparing technology basis ", fiber crops
Vertical man (Japanese) work, Chen Guorong translates, Chemical Industry Press, 2009) method in, utilizes electron beam to steam
Plating machine is sample (i.e. expose form the substrate regions of electrode and shelter the substrate of the substrate regions being formed without electrode)
On be deposited with thick Cr and 50nm thickness Au of 5nm successively, it is thus achieved that electrode, respectively as source electrode 7 and electric leakage
Pole 8 is stand-by.
(5) with reference to Fig. 7, remove the photoresist being deposited with on the sample of electrode, covered by graphene film 1
Region defines the electrical connection between electrode and graphene film 1, and the region not covered by graphene film 1 is formed
For fixing connection between electrode and substrate, the distance between source electrode 7 and drain electrode 8 is 2000nm.
(6) with reference to Fig. 8 and Fig. 9, on the substrate defining electrode, to be provided with graphene film 1 and electricity
The real estate of pole is bottom surface, and (length of side is to form culture chamber to surround sidewall (cultivating cavity wall 9) with PDMS
1.5 × 1.5cm, the degree of depth is 1cm) so that when this culture chamber seals liquid up for safekeeping, graphene film 1 is completely soaked
In liquid.
Structure is sensing element prepared by the present embodiment substantially as shown in Figure the element shown in 10.Optics is utilized to show
Micro mirror observes its microstructure, as shown in Figure 10: middle connect for Graphene (Graphene), four
Connecting line is source (or leakage) electrode, and material is chromium gold (Cr/Au).
Testing example 1
The present embodiment is used for illustrating the electric property of sensing element.
With reference to Figure 11, use the sensing element of preparation embodiment 1 preparation, by two of graphene film two ends
Cr/Au electrode is respectively as source electrode and drain electrode, and Ag/AgCl electrode is as reference electrode (ie in solution grid electricity
Pole (Vgate)), thus it is assembled into sensor.
By drain electrode ground connection, the voltage Vsd(0.01-0.1V being fixed in source electrode), measure source electrode and
Electric current Isd between drain electrode is with the variation relation of gate electrode voltage Vgate, to characterize the electronics of graphene film
Transport property.The hole of the Isd-Vgate curve shown in Figure 12 is propped up asymmetric with electronics Zhi Mingxian, dirac
The Graphene of point (minimum point) relative intrinsic occurs in that translation on Vgate axle, shows that Graphene is pasted by it
The doping impact of the Si/SiO2 substrate covered.
Testing example 2
The present embodiment is used for the electric property of sensing element being described and in the change of chemical substance in measuring solution
Application.
With reference to Figure 11, use the sensing element of preparation embodiment 1 preparation, by two of graphene film two ends
Cr/Au electrode is respectively as source electrode and drain electrode, and Ag/AgCl electrode is as reference electrode (ie in solution grid electricity
Pole (Vgate)), thus it is assembled into sensor.
Voltage is applied, simultaneously at source electrode and drain electrode (Cr/Au by the reference electrode Ag/AgCl in solution
Electrode) between apply constant channel voltage.Sample solution, sample is added in liquid storage tank (i.e. culture chamber)
In this solution, the change of charged ion concentration and kind can change the concentration of electric charges of graphenic surface, thus changes
The electric current of graphene field effect pipe.By detecting the change of Graphene electric current, chemistry in solution can be detected
The change of material.
Based on above-mentioned Cleaning Principle, graphene field effect device is used for detecting the change of pH value of solution.To cultivation
Chamber adds the pH value calibration solution of different pH value (pH is respectively 6,7,8 and 9) according to this (purchased from the U.S.
Sigma company), due to charged ion concentration and the change meeting of kind in the sample to be tested solution of different pH value
Change the concentration of electric charges of graphene membrane surface, and then change the electric current of graphene field effect pipe.As shown in figure 13,
Detect that the electric conductivity value of graphene field effect pipe occurs corresponding change, react extremely rapid and sensitive, different
Change stepped between pH value.
Embodiment 1
By applying constant channel voltage between the source electrode at graphene film two ends and drain electrode, electricity at this
Pressure graphene nanobelt produces a constant electric current.Cell or tissue is formed with graphene film in device
Being in close contact (as shown in figure 14), when the transmembrane potential of cell changes, this small voltage is to logical
Cross source electrode and electromotive force that drain electrode is applied on graphene film produces impact, and then make to flow through graphene film
Electric current produces corresponding change, by detecting this curent change and then realizing the action potential on cell membrane
The measurement of change.
Based on above-mentioned Cleaning Principle, graphene field effect device (i.e. the sensing element of embodiment 1) is used for
The change of detection nervous cell membrane potential.Reference literature Stefanie K, Gary B.Culturing hippocampal
Neurons.Nature protocol, the method in 2006,1,2406-2415, by neural for the hippocampus of mice district digested
Cell is placed in the culture chamber of graphene field effect device (having cell culture fluid DMEM in culture chamber).Warp
After the cultivation of 2 weeks, neurocyte was formed with graphene film after differentiation and contacts, as shown in figure 14.With stone
Ink alkene film contact neurocyte can the spontaneous generation signal of telecommunication, graphene field effect pipe is able to record that correspondence
The action potential of neurocyte, detected electromotive force at about 1mV, signal to noise ratio more than 5 times, as
Shown in Figure 15, the signal to noise ratio measured compared to microelectrode array sensor improves a lot.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned enforcement
Detail in mode, in the technology concept of the present invention, can enter technical scheme
The multiple simple variant of row, these simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technical characteristic described in above-mentioned detailed description of the invention,
In the case of reconcilable, can be combined by any suitable means, in order to avoid unnecessary weight
Multiple, various possible compound modes are illustrated by the present invention the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as it is not
Running counter to the thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (20)
1. detect a sensor for neurocyte electricity physiological signal, including substrate, cultivate cavity wall, graphite
Alkene film, source electrode, drain electrode and reference electrode, described substrate and culture chamber wall seamless sealing-in surround cultivation
Chamber, the substrate in described culture chamber is attached with graphene film, and described graphene film can make god through chemical modification
Grow thereon through cell, described source electrode and drain electrode electrically connect with described graphene film respectively and respectively with
Described substrate is fixing to be connected, in the region forming source electrode on substrate or the region forming drain electrode, by stone
Region and the area ratio in region not covered by graphene film that ink alkene film covers are 1:1；Described reference electrode is put
In described culture chamber.
Sensor the most according to claim 1, it is characterised in that described chemical modification is that poly relies
Propylhomoserin and/or laminin，LN are modified.
Sensor the most according to claim 2, it is characterised in that described chemical modification is that poly relies ammonia
The non-covalent bond of acid and/or laminin，LN is modified.
Sensor the most according to claim 1, it is characterised in that described substrate includes silicon chip basic unit
With silicon dioxide surface layer, described graphene film is attached on described silicon dioxide surface layer.
Sensor the most according to claim 1, it is characterised in that the Graphene of described graphene film
The number of plies is one to two layer.
Sensor the most according to claim 1, it is characterised in that the area of described graphene film is
1-200μm2。
Sensor the most according to claim 1, it is characterised in that the area of described graphene film is
10-100μm2。
Sensor the most according to claim 1, it is characterised in that the area of described graphene film is
20-30μm2。
Sensor the most according to claim 1, it is characterised in that described source electrode and drain electrode it
Between distance be 500-5000nm.
Sensor the most according to claim 1, it is characterised in that described source electrode and drain electrode it
Between distance be 750-4000nm.
11. sensors according to claim 1, it is characterised in that described source electrode and drain electrode it
Between distance be 1000-3000nm.
12. sensors according to claim 1, it is characterised in that described source electrode and drain electrode are each
From independently be metal electrode.
13. sensors according to claim 12, it is characterised in that the material of described metal electrode is
Platinum, gold and/or chromium.
14. sensors according to claim 12, it is characterised in that the thickness of described metal electrode is
0.03-0.15μm。
15. sensors according to claim 1, it is characterised in that described reference electrode is Ag/AgCl
Electrode.
16. sensors according to claim 1, it is characterised in that the material of described cultivation cavity wall is
Polydimethylsiloxane.
The sensor of the detection neurocyte electricity physiological signal described in 17. 1 kinds of any one of claim 1-16
Manufacture method, comprises the following steps:
(1) on substrate, graphene film is adhered to；
(2) on the substrate be attached to graphene film, source electrode and drain electrode, described source electrode and electric leakage are formed
Pole electrically connects with described graphene film respectively and fixes with described substrate respectively and is connected；
(3) by described be attached to graphene film and define the substrate of source electrode and drain electrode with cultivate cavity wall without
Seam to connect and surround culture chamber so that described graphene film, source electrode and drain electrode are respectively positioned on described culture chamber
In；
(4) described graphene film is chemically modified, so that neurocyte grows thereon.
18. 1 kinds of methods detecting neurocyte electricity physiological signal, comprise the following steps:
(1) at the sensor of the detection neurocyte electricity physiological signal as described in any one of claim 1-16
Culture chamber in cultured nerve cell, make neurocyte and graphene film be in close contact；
(2) between described source electrode and drain electrode, constant channel voltage is applied, when the film electricity of neurocyte
When position changes, the electric current flowing through described graphene film produces change, by detecting on described graphene film
The transmembrane potential change of neurocyte is measured in the change of electric current.
The method of 19. detection neurocyte electricity physiological signals according to claim 18, its feature exists
In, the channel voltage applied between described source electrode and drain electrode is 10-70mV.
20. methods according to claim 19, it is characterised in that between described source electrode and drain electrode
The channel voltage applied is 30-60mV.
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