CN104760922A - Ultramicro planar electrode array sensor and preparation method thereof - Google Patents
Ultramicro planar electrode array sensor and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a dual mode (two mode signals-electrophysiological signal and electrochemical signal) ultramicro planar array sensor for quantitative assay of nerve cell quantum release. An ultramicro planar electrode (0.5-5 mum) is prepared through combination of a double layer wiring design, a high precision stepping photolithography technique and a partition isolation design. According to the method, orientated nanometer modification, biological compatibility modification and specific recognition enzyme modification are combined and performed on the surface of an electrode array so as to prepare the dual mode planar ultramicro electrode array sensor. The dual mode planar ultramicro electrode array is prepared by the method combining a micro electromechanical system technique, a nanometer modification technique and a biological modification technique. Through adoption of the method, the limitations of large electrode site size (10-50 mum) of a conventional planar microelectrode and single site detection of a rodlike carbon fiber electrode are broken. The ultramicro planar electrode array prepared by the method has small electrode site and multiple recording points, does not damage nerve cells, and can simultaneously detect neurotransmitter quantum release of a plurality of nerve cells and dual mode information of an electrophysiological action potential signal in real time in situ.
Description
Technical field
The present invention relates to biology sensor, Electroanalytical Chemistry micro-nano preparing technical field, be a kind ofly can detect ultra micro sensor array of the multiple electrochemical nerve mediator signal of cellular electrophysiologicalsensor and the many synaptic sites of cell and preparation method thereof simultaneously.
Background technology
The neurotransmitter that nerve cell axons tip vesica stores is quite stable, release of emptying one's purse in units of vesica, is called as the release of quantum formula.The arrival of one-off current potential, can make the content of about 200 ~ 300 vesicas discharge.It is understand the important direct mode of Neurotransmission, mechanism of action that nerve cell quantization neurotransmitter regulator carries out real time record.The analytical technology that monitoring cell Quantum neural computing must possess hypersensitivity, resolution, ultra-small volume are asked in high selectivity, high time resolution, high-altitude.The method of traditional detection nerve cell Quantum neural computing is that patch-clamp coordinates carbon fiber electrode technology substantially, usually the data of a small amount of 1-2 passages can only be obtained, and electrode location difficulty, complex operation, cannot long term monitoring, limit the detection to cell Quantum neural computing to a great extent.
Along with the development of MEMS (MEMS) process technology, microelectrode array (microelectrode, MEA) a kind of method of long-term, multidigit point monitoring cell is provided, conventional MEA electrode diameter is at 10 ~ 50 μm, much larger than the Quantum neural computing size of vesica and cynapse, although can individual cells be navigated to, but for being positioned the Quantum neural computing detecting synaptic vesicle, still not enough in room and time resolution ratio, the spatial diversity of cell different loci release on single electrode can not be distinguished, be difficult to measure real-time Quantum neural computing signal.Therefore need to develop the less electrode of size, study to realize the monitoring of Single cell release time-space resolution and to be deep into synapse cell gap, on more profound, inquire into cell releasing mechanism.
Summary of the invention
The present invention is directed to the real-time detection of cell and synaptic vesicle Quantum neural computing micro substance, solve present problems: individual cells or vesica can only be detected, once can only detect a kind of neurotransmitter, electrode size is excessive, be difficult to detect in cynapse size; A kind of site of design preparation is little, measuring point is many, to nerve cell not damaged, the ultra micro planar electrode array sensor that can detect multiple measuring point (detecting multiple cell, various neurotransmitters) on two-dimentional yardstick simultaneously simultaneously.
According to an aspect of the present invention, which provide a kind of ultra micro planar electrode array sensor, it is characterized in that: comprise ultra micro planar electrode array and complex function rete, wherein said ultra micro planar electrode array is by dielectric base, microelectrode array, form electrode, reference electrode, contact conductor and contact, described microelectrode array comprises multiple, on a dielectric base, multiple microelectrode array can share reference electrode and to electrode in distribution; Described complex function rete comprises that nano-modified, bio-compatible is modified, specific bio-layer modifies the decorative layer combined, and its subregion is modified at ultra micro planar electrode array.
According to a further aspect of the invention, which provide a kind of preparation method of ultra micro planar electrode array sensor, it is characterized in that, comprise the steps:
Step 1, in the dielectric base through surface clean spin coating one deck photoresist, after photoetching development formed lead-in wire and microelectrode array pattern;
Step 2, sputter one deck microelectrode conductive membrane layer at microelectrode array patterned surfaces;
Step 3, employing stripping technology remove unnecessary microelectrode conductive membrane layer, leave required electrode, lead-in wire and contact, basis of formation metal electrode array;
Step 4, on underlying metal electrod-array surface by plasma enhanced chemical vapor deposition insulating barrier;
Step 5, in the first area of basic electrode array depositing insulating barrier, the ground floor domain of two-layer wiring is adopted to carry out secondary photoetching, stepping photoetching process is adopted to form ultramicroelectrode array central figure, then the method for plasma etching is adopted, expose the first ultramicroelectrode array and contact, retain the insulating barrier that all wire surfaces cover;
Step 6, depositing the second area of basic electrode array of insulating barrier, repeating step 5 etching and expose the second ultramicroelectrode array and corresponding contacts;
Step 7, employing SU-8, PDMS or polyimides preparation have on the chip of ultramicroelectrode array by common photoetching formation dam type partitioned organization.
Ultra micro planar array sensor of the present invention compares with existing bar-shaped ultramicroelectrode sensor and has following advantage:
Cell detects grow attachment on micro-planar array sensor after, attaches better, complete original position, Non-invasive detection.Without the need to location of wasting time and energy.
Ultra micro planar array sensor can detect multiple cell, the release of multiple synaptic sites vesica simultaneously.
Ultra micro planar array sensor sensor can detect various neurotransmitters.
Cell adopts the preparation of micro-nano technique at micro-planar array sensor, can prepare in batches, and performance is more homogeneous, stable.
This ultra micro planar array sensor compares with existing Array microelectrode and has following advantage:
1, ultra micro planar array sensor electrode site is less, size range 0.5 μm-5 μm, and closer to the size range of synapse cell vesica, diffusion rapidly, is beneficial to the Transient detection of vesica Quantum neural computing.
2, ultra micro planar array sensor adopts isolated area designing technique and specificity modification technique, can realize various neurotransmitters and detect.
Ultra micro planar array sensor of the present invention adopts micro electronmechanical process technology to prepare combining nano modification technique, can realize detecting in real time the harmless of cell, the original position that can be used for the various neurotransmitters of unicellular and unicellular many synaptic vesicles Quantum neural computing in multiple site detects in real time, and detect while can realizing that different stimulated is carried out to same cell, contrast detection and statistical analysis simultaneously.
This high density ultra micro planar electrode array of the present invention have area little (0.5 μm ~ 2 μm electrode site diameters), measuring point many, nerve cell not damaged, the neurotransmitter that simultaneously can detect the nerve cell vesica of nearly up to a hundred measuring points on two-dimentional yardstick are detected.Due to the highly dense characteristic of electrod-array, the spatial positional information that single nerve cell is secreted also can be determined.Carry out partitioned organization design, same sensor array can detect various neurotransmitters simultaneously.
Above-mentioned various object of the present invention, method, feature and advantage, by obtaining more detailed explanation below in conjunction with drawings and Examples.
Accompanying drawing explanation
Fig. 1 is preparation technology's flow chart of ultra micro planar electrode array sensor in the present invention;
Fig. 2 is the double-deck fabric swatch Local map of ultra micro planar electrode array sensor in the present invention;
Fig. 3 is the individual layer fabric swatch partial plan layout of ultramicroelectrode array sensor in the present invention;
Fig. 4 is the ultra micro plane electrode micrograph that in the embodiment of the present invention, platinum black is modified.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.
The ultra micro planar electrode array sensor that the present invention proposes comprises: ultramicroelectrode array and composite film, and wherein ultramicroelectrode array is by dielectric base, microelectrode array, form electrode, reference electrode, contact conductor and contact.Described dielectric base is the carrier of whole chip, in the center on dielectric base surface, distributed circular ultramicroelectrode that several are arranged in the matrix form, that be made up of conductive film material, forms microelectrode array.Described microelectrode array comprises multiple, and on a dielectric base, multiple microelectrode array can share reference electrode and to electrode in distribution.Composite film is nano-modified layer, bio-compatible decorative layer, specific bio-layer decorative layer organic assembling form, and its subregion is modified at ultramicroelectrode array surface.Ultramicroelectrode is plane is because noble metal conductive layer sputtering thickness is only 100 ~ 300nm.The electrode that this thickness is formed is flat film.Electrod-array in centre position, contact in surrounding, to electrode and reference electrode according to design in the side of electrod-array; Lead-in wire is connecting terminal and electrod-array.
Wherein, the conductive film material that described microelectrode array is selected is metal or the metallic compound of good biocompatibility; Microelectrode array comprises 60 ~ 128 microelectrodes, wherein microelectrode can be used in the detection of Electrophysiology signal according to the difference of the complex function rete of its finishing, for the microelectrode diameter 0.5 μm ~ 5 μm of Electrophysiology signal detection, neurotransmitter electrochemical signals can also be used for detect, and for applying electro photoluminescence, apply the microelectrode diameter 0.5 μm ~ 5 μm of electro photoluminescence, microelectrode pitch 5 μm ~ 200 μm.
The conductive film material of lead-in wire and contact is identical with microelectrode, thickness is greater than 300nm, ensure the pressure that its mechanical strength can be born standard electronic components and parts Elastic metal probe and caused, and ensure repeatedly cell chulture detect use cleaning after still there is good electric conductivity.
Described ultra micro planar array sensor, can detect electricity physiological signal and the electrochemical signals of nerve cell, can detect the neurotransmitter Quantum neural computing signal of the many synaptic sites of multiple nerve cell simultaneously; Single nerve cell various neurotransmitters signal can be detected simultaneously.
Dielectric substrate materials described in the present invention is made up of the one choosing in the group forming from glass, heat resistant glass, silicon chip etc.This base material hardness and thickness moderate, can splash-proofing sputtering metal layer, tolerate certain high temperature, for next step technique lays the foundation.
Fig. 1 shows preparation method's process chart of a kind of ultra micro planar electrode array sensor that the present invention proposes.As shown in Figure 1, the method comprises:
Step 1, dielectric base is carried out cleaning treatment after, substrate applies photoresist; Positive glue can be adopted, also can adopt negative glue, different photoresist can be adopted according to difference plate-making mode, adopt ordinary photolithographic process formed after photoetching development on a photoresist needed for lead-in wire and electrode, form microelectrode array pattern.
Step 2, on full wafer base material, sputter microelectrode conductive membrane layer, described microelectrode conductive membrane layer is one deck electrode metal layer, and stripping photoresist leaves required electrode, lead-in wire and contact, basis of formation metal electrode array.Electrode is made up of double-layer structure, the upper strata comprising lower floor's Seed Layer titanium (Ti) of metal or chromium (Cr) and be made up of a kind of noble metal choosing in the group forming from gold, platinum and palladium.What on insulating barrier, the titanium of pre-sputtering or chromium lower metal layer were beneficial to gold or platinum and electrode surface has effective adhesive.Precious Metals-Gold and platinum fit cooperating electrode, because their electrochemical properties in the stability of electrode surface area, electrochemical reproducibility, non-oxidizability etc. are all very good; And processing is simple, with the cementability of glass/silicon is good and conductance is high, and there is bio-compatibility.Platinum of the present invention or layer gold are as thin as 200nm ~ 500nm.
Step 3, in basic electrode array surface, by the one in plasma enhanced chemical vapor deposition (PECVD) silica, silicon nitride, silica etc. as insulating barrier, its thickness is between 250nm ~ 500nm.
Step 4, in the first area of basic electrode array depositing insulating barrier, the ground floor domain of two-layer wiring is adopted to carry out secondary photoetching, stepping photoetching process is adopted to carry out the formation (especially for 0.5 μm ~ 2 μm electrode site diameters) of ultramicroelectrode array central figure, then the method for plasma etching is adopted, expose the ultramicroelectrode array A in the first area of basic electrode array and corresponding contacts, retain the insulating barrier that all wire surfaces cover; This step object is the lead-in wire in order to use in insulating barrier covering basic electrode array, and etching exposes the ultramicroelectrode array A and corresponding contacts that are arranged in first area;
Step 5, depositing the second area of basic electrode array of insulating barrier, repeating step 4 etching and expose ultramicroelectrode array B, to electrode, reference electrode and corresponding contacts B.Wherein, two-layer wiring design can form reference electrode at ground floor, also can form reference electrode at the second layer to electrode, to electrode, and ultramicroelectrode array A and B can share reference electrode and to electrode.Two-layer wiring is ensureing, under good conductive wire scenario, to be convenient in limited plane, form more intensive ultramicroelectrode site, to be beneficial to the Quantum neural computing of catching synapse cell site.
Step 6, partitioned organization: adopt PDMS, polyimides, SU-8 etc. to form partitioned organization, isolation dam is formed with the polymeric material being highly 2 μm ~ 2000 μm between different structure, multiple electrode site is separated into multiple region, be convenient to different modifying and various neurotransmitters detection, carry out not homospecificity at regional and modify and various neurotransmitters detection.Polymeric barrier materials selects PDMS, polyimides, SU-8 one or both combining structure wherein.
Step 6, form complex function rete at electrode face finish, its modification is undertaken by following sequencing: nano-modified, specific bio-layer is modified, bio-compatible is modified, and can modify one deck in above-mentioned three layers, two-layer or three layers for zones of different as required.
Nano-modified layer: different subregion fixes nano material, in order to increase electrode performance.Nano-modified layer adopts metal or other excellent conductive material to carry out pointed decoration.Modified metal material comprises platinum, gold, titanium, rhodium, osmium etc., wherein preferred platinum black, titanium nitride.Other excellent conductive material comprises carbon, polypyrrole etc., preferred carbon nanomaterial.In order to the electrode surface plasma cleaner obtaining high-efficiency electrochemical activity carries out activation process to electrod-array.Electrode, after surface activation process, not only can remove the pollutant of electrode surface, can also play the effect on activated electrode surface simultaneously, and increase the hydrophily of electrode surface, the condition of etching can do suitable adjustment according to the situation of electrode surface.Nano-modified layer pointed decoration can adopt plating, electropolymerization, combined polymerization and micro-coating etc.
Special selection reacts decorative layer: fixing different specific materials (as biologic specificity decorative materials etc. such as structure selectivity decorative material, enzymes), detects different neurotransmitters for correspondence.Modification is assembled into ultra micro planar array sensor.Special selection reaction decorative layer comprises enzyme or antibody or antigen or macromolecule M8003 line reagent and electron acceptor, auxiliary reagent.Enzyme reagent comprises oxidizing ferment, reductase etc.; Antibody comprises monoclonal antibody, how anti-etc.; Electron acceptor comprises electron acceptor by the one selected in the group formed from ferricyanate, methylene blue, Ferrocene and its derivative and rhodium/osmium ion polymer; Auxiliary reagent is made up of coupling reagent, enzyme/antibody activator, buffer solution and surfactant etc.
Bio-compatibility selective modification, comprise the affine modification in local of ultramicroelectrode site areas, adopt the one in polylysine (polylysine), mercaptopropionic acid, polymine (PEI), laminine (laminin) or combination.Lead areas adopts PEG, Teflon etc. to carry out repelling the modification of protein substance.Wherein, nano-modified layer, special selection reaction decorative layer, bio-compatibility decorative layer are that successively order is modified.Successively can modify at the same area, also can modify respectively in zones of different.
Embodiments provide a kind of method of carrying out nano-modified layer at electrode surface.In this embodiment, undertaken nano-modified by assembling one deck nano particle at electrode surface.Concrete, by electrode surface plating platinum black or electropolymerization polypyrrole, the electrode nanometer layer with different pore size can be obtained.The method further enhances the hydrophily of electrode surface, increases the effective surface area of electrode.Not only be conducive to the immobilization of enzyme reagent, and add the electro-chemical activity of electrode.
Reaction reagent is fixed in electrode nanometer layer and forms reagent layer by the present invention, the reaction reagent be fixed in electrode nanometer layer comprises electron acceptor, react with analyte and the composite reagent with the coupled reaction of the corresponding electric current of analyte concentration can be produced, also comprising electron mediator, enzyme reagent, buffer solution and surfactant etc. simultaneously.
The reaction principle of agent combination institute of the present invention foundation is as follows:
Wherein Ach acetylcholine; AchE acetylcholine lipolytic enzyme; Acetate lactic acid; Choline choline; ChOx choline oxidase, betaine aldehyde trimethylamine acetaldehyde.
Embodiments of the invention, provide the method for fixation reaction reagent on nano particle.
First at electrode face finish osmium polymer conductive media body polymer, normal temperature places more than 12 hours.Then by after the mixing such as the acetylcholine oxidizing ferment of debita spissitudo, acetylcholine lipolytic enzyme, glutaraldehyde and bovine serum albumin(BSA), be coated in electrode surface immediately, in 35 ~ 37 DEG C of drying boxes after dry 20 ~ 25 minutes, deionized water rinsing removes uncrosslinked glutaraldehyde, after air drying, sealing cryopreservation.
The electron acceptor playing redox is by the one selected from redox polymers (Os, rhodium etc.), ferricyanate, methylene blue, Ferrocene and its derivative, the group that forms stupid quinone, phenazine methosulfate, indophenols and derivative thereof and β-Naphthoquinone-4-potassium Sulfo-nate; By electron mediator by the electronics that produces in enzyme reaction process from enzyme reaction central transference to electrode surface, the response speed of mediated enzyme biosensors and detection sensitivity are improved, reduce the voltage of reaction simultaneously.Described electron acceptor is Os redox polymers, effectively can reduce detectability, makes operating potential be down to about 0V, reduces the interference of other active material in blood.
The one selected in the group that buffer solution is phosphate buffer, TRIS buffer solution, MES buffer solution and physiological saline form; Described buffer solution is phosphate buffer.Buffer solution is for providing the reaction environment that a pH value is stable, and Optimal pH reaction is 6 ~ 8.
Described surfactant is TritonX-100.Add the surfactant of 0.01 ~ 1% nonionic, improve mixed liquor and the affinity rate of examination bar, make mix reagent more easily evenly be coated in electrode surface rapidly, the coat of formation is thin and even, electron transport rate when being beneficial to raising detection.When surfactant concentration higher than 0.5% time, enzymatic activity is suppressed and affects.Therefore 0.01 ~ 0.1% concentration surface activating agent is selected.
Above-mentioned various object of the present invention, method, feature and advantage, by obtaining more detailed explanation below in conjunction with drawings and Examples.
Fig. 1, Fig. 2 are preparation technology's flow chart and the part plan enlarged drawing of the ultramicroelectrode sensor that the present invention designs.First insulating substrate sheet glass (thickness is about 1mm) is cleaned, successively on glass, apply photoresist (positive glue 6130 or 2840 or AZ1500), common photoetching development forms electrode and lead contact figure, then noble metal electrode film Ti basalis (thick 20 ~ 30nm) and Pt (200 ~ 300nm) is sputtered, stripping photoresist, basis of formation electrod-array; PECVD depositing insulating layer Si3N4 (300 ~ 500nm); Adopt stepping photoetching process to carry out secondary photoetching (adopting the ground floor domain of two-layer wiring to carry out) in the first area of basic electrode array surface, photoresist is formed required ultramicroelectrode array figure; Adopt reactive ion etching process to carry out etching ultramicroelectrode array figure and remove insulating barrier, insulating barrier and electrode material membrane are formed ultramicroelectrode array figure A and contact (15 × 4 array of row electrodes: electrode site diameter is respectively 2 μm, 3 μm, 4 μm, 5 μm (as shown in Figure 3); Wherein less electrode is more beneficial to vesica release and detects); Repeat photoetching, noble metal sputtering at the second area of basic electrode array surface, cover insulating barrier, adopt the second layer domain of two-layer wiring to carry out secondary photoetching and plasma etching step, prepare ultramicroelectrode array B, to electrode, reference electrode and contact B (15 × 4 array of row electrodes: electrode site diameter is respectively 2 μm, 3 μm, 4 μm, 5 μm (as shown in Figure 3)).Spin coating SU-8 glue, common photoetching development, forms the electrod-array of cut zone (10 ~ 20 μm of height split dam).
Ultramicroelectrode array forms nanometer layer (as shown in Figure 4) by plating.Electroplate liquid is the pyrroles of 0.1M, the KCl of 0.1M, the plating operating potential of 0.5 ~ 0.7V is adopted to carry out constant voltage plating, or adopt the operating current of 0.01 ~ 0.05mA to carry out constant current plating, electroplating time is 30 seconds ~ 3 minutes, also-0.1V ~ 0.8V can be adopted to carry out cyclic voltammetric plating, scanning 20 ~ 40 circle.Also Au, Pt can be selected to carry out the plating of nano metal material.Then enzyme layer is modified, such as acetylcholine surveyed area: acetylcholine lipolytic enzyme 100U/ml; Choline oxidase: 100U/ml; Triton X-100:0.01%, glutaraldehyde: 0.1%, bovine serum albumin(BSA): 5%.Dopamine surveyed area then modifies 0.1 ~ 1%Nafion, dry formation Nafion film.Different transmitter detection zone modifies different enzymes or specific detection material.After modification completes, sealing Cord blood.Before using, first carry out ultraviolet sterilization, then carry out the selective modification of bio-compatibility material in electrode site district, select polylysine to carry out applying or press back.And then inoculate nerve cell and cultivate.
The present invention has used preferred embodiment to be described, and preferred embodiment is for illustrative purposes, instead of limitation of the present invention.The present invention can be many modifications and changes on the basis of the above description.Therefore, within the scope of the appended claims, the present invention can have is not other above-mentioned implementation.Such as: the difference of electrode shape, other nano particle and non-nano material modify electrode reaction district, different agent combination forms etc.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a ultra micro planar electrode array sensor, it is characterized in that: comprise ultra micro planar electrode array and complex function rete, wherein said ultra micro planar electrode array is by dielectric base, microelectrode array, form electrode, reference electrode, contact conductor and contact, described microelectrode array comprises multiple, on a dielectric base, multiple microelectrode array can share reference electrode and to electrode in distribution; Described complex function rete comprises that nano-modified, bio-compatible is modified, specific bio-layer modifies the decorative layer combined, and its subregion is modified at ultra micro planar electrode array.
2. ultra micro planar electrode array sensor as claimed in claim 1, is characterized in that: the method preparation that it adopts micro electro mechanical system (MEMS) technology, nano-modified technology and bio-modification technology to combine.
3. ultra micro planar electrode array sensor according to claim 1, is characterized in that: the conductive film material that described microelectrode array is selected is metal or the metallic compound of good biocompatibility; Microelectrode array comprises 60 ~ 128 microelectrodes, and wherein microelectrode has following difference in functionality according to the complex function rete difference of its finishing: Electrophysiology signal detection, neurotransmitter electrochemical signals detect and apply electro photoluminescence.
4. ultra micro planar electrode array sensor according to claim 1, it is characterized in that: for the microelectrode diameter 0.5 μm ~ 5 μm of Electrophysiology signal detection, detect for neurotransmitter electrochemical signals and apply the microelectrode diameter 0.5 μm ~ 5 μm of electro photoluminescence, microelectrode pitch 5 μm ~ 200 μm; The conductive film material of lead-in wire and contact is identical with microelectrode, and thickness is greater than 300nm.
5. ultra micro planar electrode array sensor according to claim 1, is characterized in that: described ultra micro planar array sensor can detect the neurotransmitter Quantum neural computing signal of the many synaptic sites of multiple nerve cell simultaneously and/or detect single nerve cell various neurotransmitters signal simultaneously.
6. a preparation method for ultra micro planar electrode array sensor, is characterized in that, comprises the steps:
Step 1, in the dielectric base through surface clean spin coating one deck photoresist, after photoetching development formed lead-in wire and microelectrode array pattern;
Step 2, sputter one deck microelectrode conductive membrane layer at microelectrode array patterned surfaces;
Step 3, employing stripping technology remove unnecessary microelectrode conductive membrane layer, leave required electrode, lead-in wire and contact, basis of formation metal electrode array;
Step 4, on underlying metal electrod-array surface by plasma enhanced chemical vapor deposition insulating barrier;
Step 5, in the first area of basic electrode array depositing insulating barrier, the ground floor domain of two-layer wiring is adopted to carry out secondary photoetching, stepping photoetching process is adopted to form ultramicroelectrode array central figure, then the method for plasma etching is adopted, expose the first ultramicroelectrode array and contact, retain the insulating barrier that all wire surfaces cover;
Step 6, depositing the second area of basic electrode array of insulating barrier, repeating step 5 etching and expose the second ultramicroelectrode array and corresponding contacts;
Step 7, employing SU-8, PDMS or polyimides preparation have on the chip of ultramicroelectrode array by common photoetching formation dam type partitioned organization.
7. method according to claim 6, it is characterized in that, reference electrode and to electrode is formed, described first ultramicroelectrode array and the public rubble electrode of the second ultramicroelectrode array and to electrode when etching exposure first ultramicroelectrode array or the second ultramicroelectrode array.
8. method according to claim 6, it is characterized in that, described dam type partitioned organization height is 2 μm ~ 2000 μm, and it is by multiple electrode site is separated into multiple region, modifies and various neurotransmitters detection to carry out not homospecificity at regional.
9. method according to claim 1, is characterized in that, described method also comprises:
Step 8, on each dam type partitioned organization modification complex function rete, it is that nano-modified, specific bio-layer is modified, bio-compatible is modified to combine and is prepared from.
10. method according to claim 9, is characterized in that: step 8 specifically comprises:
Step 81, decorated nanometer decorative layer, nano-modified layer adopts metal or other excellent conductive material to carry out pointed decoration, and its modification mode adopts plating, electropolymerization, combined polymerization and micro-coating;
Step 82, modify special selection reaction decorative layer, special selection reaction decorative layer comprises enzyme or antibody or antigen or macromolecule M8003 line reagent and electron acceptor, auxiliary reagent;
Step 83, bio-compatibility selective modification, comprise the affine modification in local of ultramicroelectrode site areas, adopt the one or more combination in polylysine, mercaptopropionic acid, polymine (PEI), laminine (laminin), lead areas adopts PEG, Teflon to carry out repelling the modification of protein substance absorption.
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