CN103901089A - Sensor for detecting nerve cell electrophysiology signal and manufacturing method and detection method of sensor - Google Patents
Sensor for detecting nerve cell electrophysiology signal and manufacturing method and detection method of sensor Download PDFInfo
- Publication number
- CN103901089A CN103901089A CN201410152771.2A CN201410152771A CN103901089A CN 103901089 A CN103901089 A CN 103901089A CN 201410152771 A CN201410152771 A CN 201410152771A CN 103901089 A CN103901089 A CN 103901089A
- Authority
- CN
- China
- Prior art keywords
- graphene film
- electrode
- substrate
- sensor
- neurocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention relates to a sensor for detecting a nerve cell electrophysiology signal and a manufacturing method and a detection method of the sensor. The sensor comprises a substrate, a culture cavity wall, a graphene membrane, a source electrode, a drain electrode and a reference electrode, wherein the substrate and the culture cavity wall are seamlessly sealed to define a culture cavity; the graphene membrane is attached to the substrate in the culture cavity; the graphene membrane is subjected to chemical modification, so that nerve cells grow on the graphene membrane; the source electrode and the drain electrode are respectively and eclectically connected with the graphene membrane and are respectively and fixedly connected with the substrate; the reference electrode is arranged in the culture cavity. When the nerve cell electrophysiology signal is detected by utilizing the sensor according to the invention, and the 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, particularly, relate to a kind of method for making of the sensor and the described sensor that detect neurocyte electricity physiological signal, and use described sensor to detect the method for neurocyte electricity physiological signal.
Background technology
Field effect transistor is a kind of by the electronic component of field effect control electric current, there are grid (gate), source electrode (source) and drain electrode (drain) three terminals, carry out by putting on the electric field that the voltage of grid produces the size of current circulating in the raceway groove of control linkage source electrode and drain electrode.The raceway groove of conventional field effect transistor is made up of heavily doped silicon.
Along with the development of nanosecond science and technology, various novel low-dimension nano materials, such as silicon nanowires (SiNW), carbon nano-tube (CNT) and Graphene (graphene) etc., caused people's extensive concern with its unique character (as surface effect, bulk effect and quantum size effect etc.).Compared with traditional material, nano material has less size, higher specific surface area, better electrical properties and better biocompatibility etc.When the raceway groove of conventional field effect transistor is substituted by nano material, formed 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 conductivity is high, an excellent physical and chemical performance such as the large and electrochemical stability of physical strength, makes it have unique application advantage in high-sensitivity detection field, has caused that people pay close attention to greatly.Graphene is the planar crystal of individual layer atomic building, each atom from the teeth outwards, the variation of external environment all, by directly affecting all carbon atoms that form Graphene, makes it extremely sensitive to the response at interface, and unique structure makes it have outstanding detection sensitivity simultaneously.Highly sensitive nitrogen monoxide (NO) the gas detection chip based on Graphene of having developed at present, has the high detection sensitivity of single NO molecule, shows that Graphene has huge potentiality as detection chip sensitive element.
The analysis of neuroelectricity signal is one of main contents of current nuroinformatics research.More conventional device is microelectrode array sensor (Micro-Electrode Array, MEA) now.MEA is made up of the microelectrode being embedded on substrate.Microelectrode is mainly by metal material, the compositions such as such as platinum, gold, titanium-nitrogen-oxygen compound and indium tin oxide.Microelectrode is arranged in array on substrate.
Although the electrical signal of neurocyte granting can be detected well with microelectrode array sensor, but the noise level obtaining is inversely proportional to the size of microelectrode size, when microelectrode size reduction is to certain size time, the level of its noise will substantially exceed the size of its signal, thereby can not differentiate real signal.Therefore detect the spatial resolution of neurocyte by the method not high.
Summary of the invention
For further improving the spatial resolution and the signal to noise ratio (S/N ratio) that detect neurocyte, the invention provides a kind of method for making of the sensor and the described sensor that detect neurocyte electricity physiological signal, and use described sensor to detect the method for neurocyte electricity physiological signal.
The present inventor makes Graphene the Graphene sensing element of graphene field effect transistor, utilize the high sensitivity of Graphene to improve the size of signal on the one hand, field effect transistor is not limited to contact resistance on the one hand in addition, it is less that detecting unit can do, and therefore can utilize sensor of the present invention further to improve the signal to noise ratio (S/N ratio) and the spatial resolution that detect neurocyte.
For realizing object of the present invention, the invention provides technical scheme:
In first aspect, the invention provides a kind of sensor that detects neurocyte electricity physiological signal, comprise substrate, culture chamber wall, graphene film, source electrode, drain electrode and contrast electrode, described substrate and the sealing-in of culture chamber wall seamless surround culture chamber, on substrate in described culture chamber, be attached with graphene film, described graphene film can make neurocyte grow thereon through chemical modification, described source electrode is electrically connected with described graphene film respectively and is fixedly connected with described substrate respectively with drain electrode, and described contrast electrode is placed in described culture chamber.
As preferred version of the present invention, described chemical modification is that poly-D-lysine and/or laminin are modified.
Preferably, described chemical modification is the non-covalent bond modification of poly-D-lysine and/or laminin.
As preferred version of the present invention, described substrate comprises silicon chip basic unit and silicon dioxide surface layer, and described graphene film is attached on described silicon dioxide surface layer.
As 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 μ m
2, for example 2 μ m
2, 5 μ m
2, 8 μ m
2, 10 μ m
2, 15 μ m
2, 20 μ m
2, 30 μ m
2, 50 μ m
2, 80 μ m
2, 100 μ m
2, 120 μ m
2, 140 μ m
2, 150 μ m
2, 175 μ m
2, 185 μ m
2, 190 μ m
2, 195 μ m
2, be preferably 10-100 μ m
2, more preferably 20-30 μ m
2.
As preferred version of the present invention, 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, be preferably 750-4000nm, more preferably 1000-3000nm.
As preferred version of the present invention, described source electrode and drain electrode are metal electrode independently of one another.
Preferably, the material of described metal electrode is platinum, gold and/or chromium, can be a kind of metal material, can be also the alloy of two or more materials.
Preferably, the thickness of described metal electrode is 0.03-0.15 μ m, for example 0.04 μ m, 0.05 μ m, 0.06 μ m, 0.08 μ m, 0.10 μ m, 0.11 μ m, 0.12 μ m, 0.13 μ m, 0.14 μ m, 0.15 μ m.
Preferably, described contrast electrode is Ag/AgCl electrode.
As preferred version of the present invention, the material of described culture chamber wall is dimethyl silicone polymer (PDMS).
In second aspect, the invention provides the method for making of the sensor of the detection neurocyte electricity physiological signal described in a kind of first aspect, comprise the following steps:
(1) on substrate, adhere to graphene film;
(2) on the substrate that has adhered to graphene film, form source electrode and drain electrode, described source electrode is electrically connected with described graphene film respectively and is fixedly connected with described substrate respectively with drain electrode;
(3) described substrate and the sealing-in of culture chamber wall seamless of having adhered to graphene film and having formed source electrode and drain electrode surrounded to culture chamber, make described graphene film, source electrode and drain electrode all be positioned at described culture chamber;
(4) described graphene film is carried out to chemical modification, so that neurocyte is grown thereon.
In the third aspect, the invention provides a kind of method that detects neurocyte electricity physiological signal, comprise the following steps:
(1) in the culture chamber of the sensor of the detection neurocyte electricity physiological signal as described in first aspect, cultivate neurocyte, make neurocyte and graphene film close contact;
(2) between described source electrode and drain electrode, apply constant channel voltage, in the time that the film potential of neurocyte changes, the electric current that flows through described graphene film changes, and the film potential of measuring neurocyte by detecting the variation of electric current on described graphene film changes.
As preferred version of the present invention, the channel voltage applying between described source electrode and drain electrode is 10-70mV, for example 12mV, 15mV, 18mV, 22mV, 25mV, 30mV, 40mV, 50mV, 55mV, 58mV, 60mV, 65mV, 68mV, be preferably 30-60mV.
Beneficial effect of the present invention is: the present invention utilizes Graphene to make the Graphene sensing element of graphene field effect transistor, because Graphene has high sensitivity, signal to noise ratio (S/N ratio) and the spatial resolution of utilizing sensor of the present invention to detect neurocyte electricity physiological signal significantly improve.Experiment confirms, utilizes the signal to noise ratio (S/N ratio) of sensor detection neurocyte electricity physiological signal of the present invention more than 5 times, and microelectrode array sensor compared to existing technology improves a lot.
Accompanying drawing explanation
Accompanying drawing is to be used to provide a further understanding of the present invention, and forms a part for instructions, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.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 square section schematic diagram of the substrate that has adhered to graphene film of the present invention.
Fig. 4 is the square section schematic diagram of the substrate of having sheltered with photoresist of the present invention.
Fig. 5 is the square section schematic diagram of the substrate after exposure of the present invention development, has shown the region that forms electrode.
Fig. 6 is the square section schematic diagram of the substrate that has formed source electrode and drain electrode of the present invention.
Fig. 7 is the square section schematic diagram that has formed source electrode and drain electrode and removed the substrate of photoresist of the present invention.
Fig. 8 is the square section schematic diagram that is provided with culture chamber and has injected the sensor of liquid in culture chamber of the present invention.
Fig. 9 is the schematic top plan view of sensor of the present invention, does not comprise contrast electrode.
Figure 10 is the optical microscope photograph of sensing element of the present invention.
Figure 11 is the square section schematic diagram of sensor of the present invention, comprises contrast electrode Ag/AgCl.
Figure 12 is electric current in the sensing element of the present invention variation diagram along with contrast electrode voltage Vgate.
Figure 13 is the relational result figure that the electric current in sensing element of the present invention changes along with the pH value difference of liquid sample.
Figure 14 is the schematic diagram that the cell membrane of sensing element of the present invention and measured neurocyte forms close contact.
Figure 15 is the result figure of the hippocampus neurocyte action potential of the mouse that arrives by sensing element senses of the present invention.
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-culture chamber wall
Embodiment
Below the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
Sensor of the present invention comprises chip unit and culture chamber wall.Wherein, chip unit comprises sensing element and contrast electrode.Described sensing element comprises substrate and is fixed on source electrode and the drain electrode on described substrate, and is electrically connected with graphene film between source electrode and drain electrode.
Wherein, described sensing element can be used as the element of field effect transistor chemical biosensor.
In preferential situation, described sensing element method as follows makes: (1) adheres to graphene film on substrate; (2) on the substrate that has adhered to graphene film, form electrode, described electrode comprises source electrode and drain electrode, and between source electrode and graphene film and between drain electrode and graphene film, is forming contact electrical connection.
Wherein, described graphene film can be the conventional graphene film using, the graphene film for example obtaining by the method for mechanical stripping, the graphene film that also can grow with chemical vapour deposition technique.
The operation of wherein, adhering to graphene film on substrate can realize by the mode that graphene film is pressed on substrate.
Wherein, can under optical microscope, position and its border of mark the graphene film on substrate based on interference effect, can also utilize Raman spectrum and atomic force microscope further to identify its number of plies.
The method according to this invention, wherein, under preferable case, in step (1), substrate comprises basic unit and is attached to 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 directly be prepared by the mode that silicon chip is oxidized, and is also easy to carry out the graphene film on substrate is positioned and its border of mark under optical microscope based on interference effect.
The method according to this invention wherein, in preferential situation, in step (2), specifically forms formation source, the region electrode of source electrode on substrate, and the region of described formation source electrode comprises the region being covered by graphene film and the region not covered by graphene film; The region that forms drain electrode on substrate forms drain electrode, and the region of described formation drain electrode comprises the region being covered by graphene film and the region not covered by graphene film; Distance between described source electrode and drain electrode is 500-5000nm, is preferably 750-4000nm, more preferably 1000-3000nm.Wherein, the distance between source electrode and drain electrode refers to the distance between immediate 2 between adjacent source electrode and drain electrode.
Wherein, under preferable case, form the region of source electrode or form in the region of drain electrode on substrate, the Area Ratio in the region being covered by graphene film and the region that do 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, its border location can be determined by observation by light microscope in the region being covered by graphene film.The boundary in the region covering at graphene film can select to comprise that the region being covered by graphene film and the region not covered by graphene film are as form the region of source electrode or the region of formation drain electrode on substrate.Be selected in behind the region of formation source on substrate (or leakage) electrode, can expose and form the substrate regions of electrode and shelter the substrate regions that does not form electrode by the mode of conventional electron beam exposure, then carry out formation source (or leakage) electrode by hot evaporation or electron beam evaporation plating.Can also after formation electrode, remove covert with organic solvent.Wherein, source electrode and drain electrode can be the conventional electrode using in field effect chemical biosensor field, it can be for example the metal electrode obtaining by hot evaporation or electron beam evaporation plating, the material of metal electrode can be at least one in platinum, gold and chromium, and the thickness of electrode can be 0.03 μ m-0.15 μ m.Source electrode and drain electrode are respectively and between substrate, be provided with for fixing being connected.The metal electrode for example obtaining by hot evaporation or electron beam evaporation plating, because the region that forms electrode has comprised the region being covered by graphene film and the region not covered by graphene film, the region being covered by graphene film has on the one hand formed being electrically connected between electrode and graphene film, and the region not covered by graphene film has on the other hand formed between electrode and substrate for fixing being connected.
The method according to this invention, wherein realizes seamless sealing-in between chip unit and culture chamber wall.One can be preferred embodiment: first dimethyl silicone polymer (PDMS) is mixed according to the ratio of mass ratio 10:1 with SYLGARD184 organosilicon body, pour 10cm into
2double dish in, make the upper surface flush of PDMS and double dish, then at 60 ℃ of curing 4-5 hours; Then the PDMS being cured is taken out from double dish, cutting therebetween a length of side is the culture chamber that 1.5 × 1.5cm, the degree of depth are 1cm.Then culture chamber is processed with oxygen plasma, processing parameter is power 10-200W, and oxygen flow is 10-150sccm, and the time is 10-120s.Then being fixed on substrate after culture chamber after treatment, guarantee that sensor unit (source electrode, drain electrode and graphene film) is in culture chamber, surrounding is carried out seamless sealing-in with PDMS again.
The method according to this invention, removes residual harmful substance the sensor chip processing with deionized water, and then sensor chip being immersed in to concentration is to carry out sterilizing in 75% ethanol, and the time is 2-10h, is preferably 3h.
The method according to this invention, grows on graphene film in order to realize neurocyte, need to modify accordingly graphene film.The example of the modification of typical case but indefiniteness such as: non-covalent bond modification is carried out in graphene film surface by least one in poly-D-lysine or laminin, wherein, the technological parameter of polylysine modification is: poly-D-lysine concentration is 1mg/ml-20mg/ml, reaction time is 1-2 hour, modifying temperature is 37 ℃, and pH value is 7.4; The modification parameter of laminin is: laminin concentration is 1mg/ml-10mg/ml, and the modification time is 1-2 hour, and modifying temperature is 37 ℃, and pH value is 7.4.After modification process, adopt phosphate (PBS) damping fluid of sterilizing to rinse.
The method of detection neurocyte electricity physiological signal provided by the invention, comprises the following steps:
(1) in the culture chamber of the sensor of detection neurocyte electricity physiological signal of the present invention, cultivate neurocyte, make neurocyte and graphene film close contact;
(2) between described source electrode and drain electrode, apply constant channel voltage, in the time that the film potential of neurocyte changes, the electric current that flows through described graphene film changes, and the film potential of measuring neurocyte by detecting the variation of electric current on described graphene film changes.
The method according to this invention, wherein, separates and cultivates the neurocyte of hippocampus.Can reference literature Stefanie K, Gary B.Culturing hippocampal neurons.Nature protocol, the method in 2006,1,2406-2415 is carried out.Separate and cultivate other neurocyte and can carry out according to technological means well known in the art.Described animal can be conventional animal used as test, as amphibian, birds or mammal, includes but not limited to toad, chicken, mouse, rat, dog, rabbit and monkey.
The method according to this invention, wherein, the voltage between source electrode and drain electrode can be 10-70mV, is preferably 30-60mV.
To describe the present invention by embodiment below.In following examples, scanning electron microscope is purchased from the model S-4800 of HIT, and the tester of electrical properties is that agents useful for same is the analytical reagent being purchased purchased from the model Axopatch200B of AXON company of U.S. electrical testing system.
Preparation Example 1
With reference to figure 1-11, the present embodiment is used for illustrating the step of preparing sensing element in the method according to this invention, prepares the step of deflocculated graphite alkene field effect device.
(1) with reference to figure 1, according to document Li X S, Cai W W, An J H, et al.Large-area synthesisof high-quality and uniform graphene films on copper foils.Science, in copper substrate, (size is 4cm × 8cm to the method for method in 2009,324:1312 – 1314 by chemical gaseous phase deposition, and thickness is that 100 μ m) go up growing graphenes.Spin coating one deck 300nm is thick on the copper sheet that has Graphene photoresist (PMMA950K, purchased from German ALLRESIST company), baking is volatilized solvent completely at 120 ℃; Having the copper sheet of photoresist to put into copper etching liquid (containing the solution of 1mol/L ferric trichloride and 1mol/L hydrochloric acid) spin coating is etched to copper and reacts completely; Obtain being attached to the graphene film on photoresist.
(2) with reference to figure 1-3, by the silicon chip (size for 3cm × 3cm, thickness is 0.52cm, purchased from U.S. sillicon Valley Microelectrics) with the thick thermal oxide SiO2 of 280nm as substrate, be that basic unit 3 is silicon chip, the substrate that surface layer 2 is silicon dioxide layer.The graphene film being attached on photoresist is transferred on substrate, and pressed and make graphene film and substrate close contact; Then by photoresist lysate (acetone), the photoresist on graphene film surface is dissolved, retain graphene film on substrate, obtain being attached with the substrate (Fig. 3) of graphene film 1.Located under optical microscope based on interference effect and mark boundaries, for determining the region that forms electrode.
(3), with reference to figure 4 and Fig. 5, on the substrate that is attached with graphene film 1, form source electrode and drain electrode.Particularly, revolve the photoresist that Tu one deck 300nm is thick (PMMA950K, purchased from German ALLRESIST company) at the substrate surface that is attached with graphene film 1, be baked to solvent evaporates at 180 ℃ complete.According to the border of the graphene film 1 of mark under optical microscope, determine the region 5 of formation source electrode and the region 6 of formation drain electrode, the Area Ratio in the region being covered by graphene film 1 in the region of formation source (or leakage) electrode (width is the strip electrode of 2 μ m) and the region not covered by graphene film 1 is 1:1, exposed in the region of formation source (or leak) electrode with electron beam exposure apparatus, development 10min in developer solution (4-methyl-2 pentanone and isopropyl alcohol are pressed the mixed liquor of the volume ratio mixing of 1:5), in isopropyl alcohol after photographic fixing 10min, dry up with nitrogen; Expose thus the substrate regions of formation electrode and shelter the substrate regions that does not form electrode.
(4) with reference to figure 6, under high vacuum condition, according to document (" film preparing technology basis "; the vertical man of numb Dill (Japan) is outstanding, and Chen Guorong translates, Chemical Industry Press, 2009) method in, utilize electron beam evaporation plating machine evaporation 5nm is thick successively on sample (exposing the substrate regions that forms electrode and the substrate of sheltering the substrate regions that does not form electrode) Cr and the thick Au of 50nm, obtain electrode, stand-by as source electrode 7 and drain electrode 8 respectively.
(5) with reference to figure 7, photoresist on the sample of the good electrode of removal evaporation, the region being covered by graphene film 1 has formed being electrically connected between electrode and graphene film 1, the region not covered by graphene film 1 has formed between electrode and substrate for fixing being connected, and the distance between source electrode 7 and drain electrode 8 is 2000nm.
(6) with reference to figure 8 and Fig. 9, forming on the substrate of electrode, take the real estate that is provided with graphene film 1 and electrode as bottom surface, (length of side is as 1.5 × 1.5cm to form culture chamber to surround sidewall (culture chamber wall 9) with PDMS, the degree of depth is 1cm), so that in the time that this culture chamber is sealed liquid up for safekeeping, graphene film 1 is soaked in liquid completely.
Structure substantially element is as shown in figure 10 sensing element prepared by the present embodiment.Utilize its micromechanism of observation by light microscope, as shown in figure 10: that centre connects is Graphene (Graphene), four connecting lines are source (or leakage) electrode, and material is chromium gold (Cr/Au).
Test implementation example 1
The present embodiment is used for illustrating the electric property of sensing element.
With reference to Figure 11, the sensing element that uses Preparation Example 1 to prepare, using two Cr/Au electrodes at graphene film two ends, respectively as source electrode and drain electrode, Ag/AgCl electrode is as contrast electrode (ie in solution gate electrode (Vgate)), thereby is assembled into sensor.
By drain electrode ground connection, the voltage Vsd(0.01-0.1V being fixed on the electrode of source), the electric current I sd between measurement source electrode and drain electrode is with the variation relation of gate electrode voltage Vgate, to characterize the Electronic Transport Properties of graphene film.The hole of the Isd-Vgate curve shown in Figure 12 is propped up with electronics Zhi Mingxian asymmetric, and dirac point (minimum point) the relatively Graphene of intrinsic has occurred translation on Vgate axle, shows that Graphene is subject to the doping impact of its Si/SiO2 substrate pasting.
Test implementation example 2
The present embodiment is used for illustrating the electric property of sensing element and the application in the variation of measuring chemical substance in solution.
With reference to Figure 11, the sensing element that uses Preparation Example 1 to prepare, using two Cr/Au electrodes at graphene film two ends, respectively as source electrode and drain electrode, Ag/AgCl electrode is as contrast electrode (ie in solution gate electrode (Vgate)), thereby is assembled into sensor.
Apply voltage by the contrast electrode Ag/AgCl in solution, between source electrode and drain electrode (Cr/Au electrode), apply constant channel voltage simultaneously.In liquid storage tank (being culture chamber), add sample solution, in sample solution, the variation of charged ion concentration and kind can change the concentration of electric charges on Graphene surface, thereby changes the electric current of graphene field effect pipe.By detecting the variation of Graphene electric current, the variation of chemical substance in solution can be detected.
Based on above-mentioned detection principle, the variation by graphene field effect device for detection of pH value of solution.To the pH value calibration liquid (purchased from Sigma company of the U.S.) that adds according to this different pH values (pH is respectively 6,7,8 and 9) in culture chamber, because the variation of charged ion concentration and kind in the sample to be tested solution of different pH values can change the concentration of electric charges on graphene film surface, and then change the electric current of graphene field effect pipe.As shown in figure 13, detect that corresponding variation occurs the electric conductivity value of graphene field effect pipe, react very rapid and sensitive, between different pH values, change stepped.
By applying constant channel voltage between the source electrode at graphene film two ends and drain electrode, under this voltage, in graphene nanobelt, produce a constant electric current.In cell or tissue and device, graphene film forms close contact (as shown in figure 14), in the time that the film potential of cell changes, this small voltage exerts an influence to the electromotive force being applied on graphene film by source electrode and drain electrode, and then make the electric current that flows through graphene film produce corresponding variation, by detecting this curent change and then realizing the measurement that the action potential in cell membrane changes.
Based on above-mentioned detection principle, the variation by graphene field effect device (being the sensing element of embodiment 1) for detection of nervous cell membrane potential.Reference literature Stefanie K, Gary B.Culturing hippocampalneurons.Nature protocol, 2006,1, method in 2406-2415, is placed in the hippocampus of mice district neurocyte having digested the culture chamber (culture chamber has cell culture fluid DMEM) of graphene field effect device.Through the cultivation of 2 weeks, neurocyte formed and contacts with graphene film after differentiation, as shown in figure 14.The neurocyte contacting with graphene film can spontaneous generation electric signal, graphene field effect pipe can be recorded to the action potential of corresponding neurocyte, detected electromotive force is in 1mV left and right, signal to noise ratio (S/N ratio) is more than 5 times, as shown in figure 15, the signal to noise ratio (S/N ratio) measuring than microelectrode array sensor improves a lot.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characterictic described in above-mentioned embodiment, in reconcilable situation, can combine by any suitable mode, for fear of unnecessary repetition, the present invention is to the explanation no longer separately of various possible array modes.
In addition, also can carry out combination in any between various embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. one kind is detected the sensor of neurocyte electricity physiological signal, comprise substrate, culture chamber wall, graphene film, source electrode, drain electrode and contrast electrode, described substrate and the sealing-in of culture chamber wall seamless surround culture chamber, on substrate in described culture chamber, be attached with graphene film, described graphene film can make neurocyte grow thereon through chemical modification, described source electrode is electrically connected with described graphene film respectively and is fixedly connected with described substrate respectively with drain electrode, and described contrast electrode is placed in described culture chamber.
2. sensor according to claim 1, is characterized in that, described chemical modification is that poly-D-lysine and/or laminin are modified;
Preferably, described chemical modification is the non-covalent bond modification of poly-D-lysine and/or laminin.
3. sensor according to claim 1 and 2, is characterized in that, described substrate comprises silicon chip basic unit and silicon dioxide surface layer, and described graphene film is attached on described silicon dioxide surface layer.
4. according to the sensor described in claim 1-3 any one, it is characterized in that, the Graphene number of plies of described graphene film is one to two layer;
Preferably, the area of described graphene film is 1-200 μ m
2, be preferably 10-100 μ m
2, more preferably 20-30 μ m
2.
5. according to the sensor described in claim 1-4 any one, it is characterized in that, the distance between described source electrode and drain electrode is 500-5000nm, is preferably 750-4000nm, more preferably 1000-3000nm.
6. according to the sensor described in claim 1-5 any one, it is characterized in that, described source electrode and drain electrode are metal electrode independently of one another;
Preferably, the material of described metal electrode is platinum, gold and/or chromium;
Preferably, the thickness of described metal electrode is 0.03-0.15 μ m;
Preferably, described contrast electrode is Ag/AgCl electrode.
7. according to the sensor described in claim 1-6 any one, it is characterized in that, the material of described culture chamber wall is dimethyl silicone polymer.
8. a method for making for the sensor of the detection neurocyte electricity physiological signal described in claim 1-7 any one, comprises the following steps:
(1) on substrate, adhere to graphene film;
(2) on the substrate that has adhered to graphene film, form source electrode and drain electrode, described source electrode is electrically connected with described graphene film respectively and is fixedly connected with described substrate respectively with drain electrode;
(3) described substrate and the sealing-in of culture chamber wall seamless of having adhered to graphene film and having formed source electrode and drain electrode surrounded to culture chamber, make described graphene film, source electrode and drain electrode all be positioned at described culture chamber;
(4) described graphene film is carried out to chemical modification, so that neurocyte is grown thereon.
9. a method that detects neurocyte electricity physiological signal, comprises the following steps:
(1) in the culture chamber of the sensor of the detection neurocyte electricity physiological signal as described in claim 1-7 any one, cultivate neurocyte, make neurocyte and graphene film close contact;
(2) between described source electrode and drain electrode, apply constant channel voltage, in the time that the film potential of neurocyte changes, the electric current that flows through described graphene film changes, and the film potential of measuring neurocyte by detecting the variation of electric current on described graphene film changes.
10. the method for detection neurocyte electricity physiological signal according to claim 9, is characterized in that, the channel voltage applying between described source electrode and drain electrode is 10-70mV, is preferably 30-60mV.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410152771.2A CN103901089B (en) | 2014-04-16 | 2014-04-16 | The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410152771.2A CN103901089B (en) | 2014-04-16 | 2014-04-16 | The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103901089A true CN103901089A (en) | 2014-07-02 |
| CN103901089B CN103901089B (en) | 2016-08-24 |
Family
ID=50992550
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410152771.2A Active CN103901089B (en) | 2014-04-16 | 2014-04-16 | The detection sensor of neurocyte electricity physiological signal and manufacture method and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103901089B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104132965A (en) * | 2014-07-11 | 2014-11-05 | 江西科技师范大学 | Experiment method for studying effect of tenuigenin on synaptic transmission of rat hippocampal neuron |
| CN105651845A (en) * | 2015-12-28 | 2016-06-08 | 中国科学院上海微系统与信息技术研究所 | Non-covalently modified graphene field effect transistor-based tumor marker detection sensor and production method thereof |
| CN106335234A (en) * | 2016-08-19 | 2017-01-18 | 中国科学院上海微系统与信息技术研究所 | Graphene and protein composite film based on non-covalent modification and preparation method thereof |
| CN107121479A (en) * | 2017-05-17 | 2017-09-01 | 中国科学院长春应用化学研究所 | A kind of electro-chemical systems |
| CN107167608A (en) * | 2017-04-06 | 2017-09-15 | 中国科学院上海微系统与信息技术研究所 | A kind of graphene tumor marker sensor based on multifunctional nano level protein films and preparation method thereof |
| CN109613071A (en) * | 2019-01-11 | 2019-04-12 | 电子科技大学 | The humidity sensor and preparation method thereof of wet sensitive composite membrane based on polylysine modification carbon-based material |
| CN111289819A (en) * | 2020-04-09 | 2020-06-16 | 中山大学 | Integrated recording regulation and control system for measuring intracellular electric signals by myocardial cell electroporation |
| CN113008967A (en) * | 2020-06-29 | 2021-06-22 | 山东大学 | Unmarked graphene field effect transistor flexible biosensor and preparation method and application thereof |
| CN113060700A (en) * | 2021-03-22 | 2021-07-02 | 清华大学 | Nano-channel high-precision processing method based on graphene anisotropic etching principle and application thereof |
| CN113176247A (en) * | 2015-10-07 | 2021-07-27 | 加利福尼亚大学校董会 | Graphene-based multi-modal sensor |
| CN113366653A (en) * | 2018-11-06 | 2021-09-07 | 西班牙高等科研理事会 | Graphene transistor system for measuring electrophysiological signals |
| CN115616058A (en) * | 2021-07-16 | 2023-01-17 | 德州奥格锐生生物科技有限公司 | Graphene sensor, manufacturing method thereof and real-time in-situ detection method for hepatocyte differentiation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1476536A (en) * | 2000-10-02 | 2004-02-18 | ���ư������ѧ����˾ | System for electro physiological measurements |
| CN1554942A (en) * | 2003-12-25 | 2004-12-15 | 浙江大学 | Single cell sensor for ectocytic action potential detection and its preparing method |
| US20050156584A1 (en) * | 2004-01-21 | 2005-07-21 | Rosemount Analytical Inc. | Ion sensitive field effect transistor (ISFET) sensor with improved gate configuration |
| CN101629945A (en) * | 2009-08-06 | 2010-01-20 | 浙江大学 | Device for detecting electrical signal of nerve cell |
| CN101639474A (en) * | 2009-08-21 | 2010-02-03 | 浙江大学 | Device for detecting three-dimensional growth of nerve cells and electric signals thereof |
| CN103430342A (en) * | 2010-12-27 | 2013-12-04 | E.T.C.有限责任公司 | Platform comprising organic field-effect transistor for biological and medical application |
-
2014
- 2014-04-16 CN CN201410152771.2A patent/CN103901089B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1476536A (en) * | 2000-10-02 | 2004-02-18 | ���ư������ѧ����˾ | System for electro physiological measurements |
| CN1554942A (en) * | 2003-12-25 | 2004-12-15 | 浙江大学 | Single cell sensor for ectocytic action potential detection and its preparing method |
| US20050156584A1 (en) * | 2004-01-21 | 2005-07-21 | Rosemount Analytical Inc. | Ion sensitive field effect transistor (ISFET) sensor with improved gate configuration |
| CN101629945A (en) * | 2009-08-06 | 2010-01-20 | 浙江大学 | Device for detecting electrical signal of nerve cell |
| CN101639474A (en) * | 2009-08-21 | 2010-02-03 | 浙江大学 | Device for detecting three-dimensional growth of nerve cells and electric signals thereof |
| CN103430342A (en) * | 2010-12-27 | 2013-12-04 | E.T.C.有限责任公司 | Platform comprising organic field-effect transistor for biological and medical application |
Non-Patent Citations (3)
| Title |
|---|
| C.-K. YEUNG ET AL.: "Validation of the use of field effect transistors for extracellular signal recording in pharmacological bioassays", 《JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS》, vol. 45, 31 December 2001 (2001-12-31) * |
| FERNANDO PATOLSKY ET AL.: "Detection, Stimulation, and Inhibition of Neuronal Signals with High-Density Nanowire Transistor Arrays", 《SCIENCE》, 25 August 2006 (2006-08-25) * |
| TZAHI COHEN-KARNI ET AL.: "Graphene and Nanowire Transistors for Cellular Interfaces and Electrical Recording", 《NANO LETT.》, 31 December 2010 (2010-12-31) * |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104132965A (en) * | 2014-07-11 | 2014-11-05 | 江西科技师范大学 | Experiment method for studying effect of tenuigenin on synaptic transmission of rat hippocampal neuron |
| CN113176247A (en) * | 2015-10-07 | 2021-07-27 | 加利福尼亚大学校董会 | Graphene-based multi-modal sensor |
| CN105651845A (en) * | 2015-12-28 | 2016-06-08 | 中国科学院上海微系统与信息技术研究所 | Non-covalently modified graphene field effect transistor-based tumor marker detection sensor and production method thereof |
| CN106335234A (en) * | 2016-08-19 | 2017-01-18 | 中国科学院上海微系统与信息技术研究所 | Graphene and protein composite film based on non-covalent modification and preparation method thereof |
| CN107167608A (en) * | 2017-04-06 | 2017-09-15 | 中国科学院上海微系统与信息技术研究所 | A kind of graphene tumor marker sensor based on multifunctional nano level protein films and preparation method thereof |
| CN107167608B (en) * | 2017-04-06 | 2019-03-19 | 中国科学院上海微系统与信息技术研究所 | A kind of graphene tumor marker sensor and preparation method thereof based on multifunctional nano grade protein films |
| CN107121479A (en) * | 2017-05-17 | 2017-09-01 | 中国科学院长春应用化学研究所 | A kind of electro-chemical systems |
| CN113366653A (en) * | 2018-11-06 | 2021-09-07 | 西班牙高等科研理事会 | Graphene transistor system for measuring electrophysiological signals |
| CN109613071B (en) * | 2019-01-11 | 2021-07-27 | 电子科技大学 | Humidity sensor of humidity-sensitive composite membrane based on polylysine modified carbon-based material and preparation method thereof |
| CN109613071A (en) * | 2019-01-11 | 2019-04-12 | 电子科技大学 | The humidity sensor and preparation method thereof of wet sensitive composite membrane based on polylysine modification carbon-based material |
| CN111289819A (en) * | 2020-04-09 | 2020-06-16 | 中山大学 | Integrated recording regulation and control system for measuring intracellular electric signals by myocardial cell electroporation |
| CN111289819B (en) * | 2020-04-09 | 2022-04-12 | 中山大学 | Integrated recording regulation and control system for measuring intracellular electric signals by myocardial cell electroporation |
| CN113008967A (en) * | 2020-06-29 | 2021-06-22 | 山东大学 | Unmarked graphene field effect transistor flexible biosensor and preparation method and application thereof |
| CN113060700A (en) * | 2021-03-22 | 2021-07-02 | 清华大学 | Nano-channel high-precision processing method based on graphene anisotropic etching principle and application thereof |
| CN113060700B (en) * | 2021-03-22 | 2023-11-21 | 清华大学 | High-precision nano-channel processing method based on graphene anisotropic etching principle and application thereof |
| CN115616058A (en) * | 2021-07-16 | 2023-01-17 | 德州奥格锐生生物科技有限公司 | Graphene sensor, manufacturing method thereof and real-time in-situ detection method for hepatocyte differentiation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103901089B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103901089A (en) | Sensor for detecting nerve cell electrophysiology signal and manufacturing method and detection method of sensor | |
| Nia et al. | Electrodeposition of copper oxide/polypyrrole/reduced graphene oxide as a nonenzymatic glucose biosensor | |
| Niu et al. | A novel electrochemical sensor of bisphenol A based on stacked graphene nanofibers/gold nanoparticles composite modified glassy carbon electrode | |
| Tang et al. | Highly sensitive dopamine biosensors based on organic electrochemical transistors | |
| Chen et al. | Bismuth-based porous screen-printed carbon electrode with enhanced sensitivity for trace heavy metal detection by stripping voltammetry | |
| Du et al. | Graphene microelectrode arrays for neural activity detection | |
| Heller et al. | Influence of electrolyte composition on liquid-gated carbon nanotube and graphene transistors | |
| Hassan et al. | High temperature SU-8 pyrolysis for fabrication of carbon electrodes | |
| Moon et al. | A nanowire-based label-free immunosensor: direct incorporation of a PSA antibody in electropolymerized polypyrrole | |
| CN104237357A (en) | Sensing element, preparation method and sensor | |
| Tian et al. | Fabrication of graphene quantum dots/silicon nanowires nanohybrids for photoelectrochemical detection of microcystin-LR | |
| Hüske et al. | Redox cycling in nanoporous electrochemical devices | |
| Stoop et al. | Charge noise in organic electrochemical transistors | |
| Qi et al. | ZnO nano-array-based EGFET biosensor for glucose detection | |
| Wang et al. | Silk fibroin hydrogel encapsulated graphene filed-effect transistors as enzyme-based biosensors | |
| CN108872346A (en) | A kind of bacteriolyze enzyme sensor based on liquid grid structure grapheme transistor and preparation method thereof and the application in lysozyme detection | |
| Neubert et al. | Faradaic effects in electrochemically gated graphene sensors in the presence of redox active molecules | |
| US20220404301A1 (en) | Three-dimensional hydrogel-graphene-based biosensor and preparation method thereof | |
| Truong et al. | Reduced graphene oxide field-effect transistor with indium tin oxide extended gate for proton sensing | |
| JP5903872B2 (en) | Transistor type sensor and method for manufacturing transistor type sensor | |
| Zhang et al. | A real-time-range potentiostat coupled to nano-Au-modified microband electrode array for high-speed stripping determination of human blood lead | |
| US11536721B2 (en) | Electrochemical immunosensors | |
| CN104237308B (en) | A kind of method screening medicine in vitro | |
| CN107037108B (en) | Using MoS2The method of film F ET detection glucose concentration | |
| Lee et al. | Band-type microelectrodes for amperometric immunoassays |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |