CN1945300A - Electrochemical ultramicro electrode combination method and its ultramicro combined electrode and preparation process - Google Patents

Abstract

The invention provides a combination method, a structure and a preparation process of an electrochemical ultramicro electrode, wherein an ultramicro working electrode and a contra/quasi-reference electrode are combined and integrated to form a two-electrode system, the two electrodes are isolated by an insulating layer with nanometer thickness, so that electrochemically reversible mass points are oxidized or reduced on one electrode after diffusion layers on the two electrodes are superposed, and are regenerated on the other electrode through reduction or oxidation and then are diffused back to the original electrode, thereby amplifying a current signal output on the electrode for detecting cells. The invention can obviously reduce the solution resistance, greatly improve the available sweeping speed and the detection sensitivity of the analysis of the actual system, reduce the damage to the research system to the minimum, has simple and convenient manufacturing of the combined electrode, long service life and easy updating, is a high-performance electrochemical biosensor, can be used for high-space-time resolution dynamic detection of single cell release and real-time dynamic analysis research of single vesicle in the cell.

Description

Electrochemical ultramicro electrode combination method and its ultramicro combined electrode and preparation process

【Technical field】

The invention relates to the technical fields of electroanalytical chemistry and biosensor, in particular to an ultramicro electrode combination method, an ultramicro combined electrode structure made according to the method, and a process method for making the electrode.

【Background technique】

Cell is the basic unit of biology, the starting point of evolution, and the basic object that life science must understand. Develop new technologies for intracellular detection, to detect neurotransmitters such as dopamine (Dopamine, DA), nitric oxide (NO), hydrogen peroxide (H ₂ O ₂ ) and The in vivo, real-time and dynamic chemical detection of information molecules such as oxygen free radicals is a very challenging frontier topic in analytical chemistry, and it is also an important entry point for in-depth research on the functions of the brain and nervous system.

The detection techniques of intracellular neurotransmitters and information molecules have been extensively studied, among which fluorescence histochemistry and immunohistology can only indirectly measure neurotransmitters, and for some free radicals with high activity, short life and extremely unstable It is difficult to measure, let alone real-time detection in cells. Electrochemical voltammetry has the characteristics of time resolution and space resolution, which provides the necessary basis for in vivo, real-time and dynamic analysis, and has become the main method for studying central nervous activity. The application ability of voltammetry depends on the upper limit of the scan rate. The higher the scan rate or frequency, the lower the kinetic time window that can be resolved will be extended to the lower end, and the faster heterogeneous electron transfer process and its coupling can be studied. Homogeneous chemical processes, tracking shorter-lived intermediates, greatly expands understanding of electron transfer and chemical reaction kinetics. Since the solution resistance R _s in a physiological system is much greater than that of an ideal system, the available scan rate of ultrafast voltammetry in a physiological environment rarely exceeds several kV/s, which is far less than the highest scan rate in an ideal system. Although R _s can be compensated by positive feedback techniques, the larger R _s , the larger the feedback resistor and compensation resistor used, resulting in reduced instrument and circuit bandwidth, making it ultimately unusable for ultrafast voltammetric analysis. Conventional intracellular voltammetry detection is to insert the ultra-micro working electrode into the cell to be tested, and place the counter electrode and reference electrode far outside the cell so as not to affect the operation. This detection method crosses the cell membrane, not only the solution resistance is relatively high Large, the stimulus and disturbance to the cell system are also great, which directly affects the reliability and sensitivity of the measurement results.

At present, the ordinary working electrode is large in size, and the disturbance and damage of the system to be measured are relatively large. It is impossible to establish an electrode system with low solution resistance in the ultrafast voltammetric research in vivo and on-site, which leads to the reduction of the bandwidth of the instrument and the circuit. The detection sensitivity is reduced, and ultimately affects the credibility of the detection results. The Chinese Patent Office discloses a preparation process of a composite microelectrode (application number: 03137469.7), which provides a method for making a miniature reference electrode in a composite microelectrode, which uses a glass capillary to The silver wire, platinum wire and carbon fiber are insulated and packaged in the inner cavity of a thin metal tube. Among them, the two ends of the carbon fiber electrode glass capillary are sealed with solid paraffin; the miniature silver/silver chloride (Ag/AgCl) miniature reference electrode uses glass The capillary phenomenon of the capillary, a section of potassium chloride saturated solution saturated with silver chloride is sealed in the capillary; the two ends of the platinum wire counter electrode glass capillary are sealed with melted solid paraffin. However, the above-mentioned method adopts a three-level electrode system, and the composite microelectrode obtained according to this method is only provided with a relatively stable reference potential by a micro-reference electrode in cell measurement, although the measurement accuracy is improved to a certain extent. However, _{the prepared} composite microelectrodes cannot adapt to and meet the requirements of in vivo, real-time and Dynamic chemical detection requirements.

Therefore, in the in vivo and on-site ultrafast voltammetry research, a new microelectrode system with low solution resistance is established to reduce the interference to the life activities of cells, so as to improve the usable scanning speed and detection sensitivity in the actual analysis environment. It is very necessary and important.

【Content of invention】

The technical problem to be solved by the present invention is to overcome the defects of the above-mentioned existing common electrode system detection that the solution resistance is relatively large, and the stimulation and disturbance to the cell system are large, which affect the reliability and sensitivity of the measurement results, and provide a A method for combining ultrafine electrodes with a wide range of applications and small volume, and the ultrafine combined electrodes obtained by the method and the preparation process of the ultrafine combined electrodes can significantly reduce the solution resistance and greatly improve the analysis efficiency of the actual system. The scan speed and detection sensitivity can be used to minimize the damage to the research system.

For realizing above-mentioned purpose of the invention, the technical scheme that the present invention proposes is:

A method for combining electrochemical ultramicro electrodes, characterized in that: it is to combine ultramicro working electrodes and alignment/quasi-reference electrodes together to form a two-electrode system, and the ultramicro-working electrodes and alignment/quasi-reference electrodes The ratio electrodes are separated by a nanometer-thick insulating layer, so that the diffusion layers on the two electrodes overlap, and the electrochemically reversible particles are oxidized or reduced on one electrode, and regenerated by reduction or oxidation on the other electrode, and then return to the electrode. Diffusion to the original electrode, so that the current signal output on the electrode of the detection cell is amplified.

An electrochemical ultramicro combined electrode structure is characterized in that: it comprises an ultramicro working electrode, an alignment/quasi reference electrode and a glass capillary, one end of the ultramicro working electrode is sealed and fixed in the glass capillary, and its end point is connected through a conductive The other end of the ultramicro working electrode is uniformly coated or polymerized with an insulating layer with a thickness of nanometer, and the insulating layer and the outer surface of the glass capillary are evenly coated with a metal layer, and the metal layer is drawn out by another wire , constituting the alignment/quasi-reference electrode.

In the above structure, the ultramicro working electrode is a carbon base electrode or a metal ultramicro electrode, wherein the carbon base electrode material can be preferably carbon fiber or carbon nanomaterial, and the metal ultramicro electrode material is preferably gold or platinum metal;

The insulating layer is made of insulating varnish, epoxy resin or polymer;

The alignment/quasi-reference electrode is made of gold, platinum, silver noble metal material or rare metal material.

The present invention also provides a preparation process for the above-mentioned electrochemical ultrafine composite electrode, which is characterized in that it includes the following specific steps:

First, bond one end of the ultramicro working electrode to the wire through conductive glue, then seal and fix the end in the capillary, and the wire is exposed outside the capillary; then evenly coat or polymerize a nanometer on the unsealed surface of the other end of the ultramicro working electrode thickness of the insulating layer, and then evenly coat a metal layer on the insulating layer and the outer surface of the glass capillary, lead it out with a wire to form an alignment/alignment reference electrode, and finally cut off the front end of the electrode coated with the metal layer and the insulating layer vertically , the exposed section of the ultramicro working electrode is used as an ultramicro composite electrode.

In the above method, the coating process of the insulating layer is to immerse the ultrafine working electrode in insulating varnish or epoxy resin, take it out and dry it after 1 to 5 minutes, and control the thickness of the insulating layer between 100-500nm;

The polymerization of the insulating layer is to immerse the ultrafine working electrode in phenol and 2-allylphenol solution, apply a voltage of 2V to 5V on the ultrafine working electrode, so that the above two chemical substances are polymerized, and the ultrafine working electrode A polymer insulating layer is formed on the surface, and the thickness of the insulating layer is controlled between 10-50nm;

The metal layer coating adopts evaporation or sputtering method to plate a layer of gold, platinum, silver metal or rare metal on the insulating layer and the outer wall of the capillary, and the thickness of the metal layer is controlled between 50-80nm.

As a further step of its preparation process, in the present invention, before the ultramicro working electrode is bonded to the wire and sealed in the capillary, the ultramicro working electrode made of carbon material is ultrasonically cleaned and dried with acetone, ethanol, double distilled water or a mixture thereof, The ultramicro working electrodes made of gold or platinum metal materials are cleaned with nitric acid and aqua regia and dried.

Compared with the prior art, the present invention has the following technical effects:

(1) The present invention integrates the two-electrode system by combining the ultramicro working electrode and the alignment/quasi-reference electrode, and the ultra-micro-working electrode and the alignment/quasi-reference electrode are separated by an insulating layer with a nanometer thickness, thus constituted The ultra-micro-combined electrode is small in size, and the working electrode and the alignment/quasi-reference electrode can be placed in the system to be tested at the same time, so it can greatly reduce the disturbance to the cell life activity system and minimize the damage to the system;

(2) In the present invention, due to the superposition of the diffusion layer on the ultramicro-combined electrode, the electrochemically reversible particle will generate a redox regeneration cycle, which can amplify the output current signal on the electrode of the detection cell, thereby greatly improving the detection sensitivity. ;

(3) The ultramicro composite electrode formed by the present invention reduces the solution resistance to a large extent, and the used feedback resistance and compensation resistance will also be correspondingly reduced, thereby not affecting the bandwidth of the instrument and the circuit;

(4) The ultrafine composite electrode of the present invention can greatly improve the available scan rate of actual system analysis, can capture instantaneous dynamic information, and is conducive to in-depth research on electron transfer and chemical reaction kinetics of the analysis system;

(5) The ultra-micro composite electrode of the present invention has a long service life, and the electrode can be updated by vertically cutting off the front end of the electrode to expose a fresh working electrode, with simple operation and good stability;

(6) The preparation process of the ultra-micro composite electrode of the present invention is simple and practical, easy to operate, easy to control the production conditions, low in cost, can be produced in general chemical laboratories, and has good popularization and application value.

The ultramicro composite electrode provided by the invention can significantly reduce the solution resistance, greatly improve the available scanning speed and detection sensitivity in the analysis of the actual system, and minimize the damage to the research system. It is a high-performance electrochemical biosensor , which can be used for high-spatial-resolution dynamic detection of single-cell release and real-time dynamic analysis of single vesicles in cells.

【Description of drawings】

Fig. 1 is the structural end face and the side schematic diagram of the electrochemical ultramicro composite electrode of the present invention;

Fig. 2 is the cyclic voltammogram of 12mmol/L anthracene in acetonitrile solution with 0.1mol/L tetraethylammonium tetrafluoroborate (NEt ₄ BF ₄ ) supporting electrolyte when the sweep rate is 1.34MV/s;

Figure 3 is the differential pulse voltammogram of 10 μmol/L DA in 0.1mol/L phosphate buffer solution on the gold/platinum ultramicro composite electrode when the scan rate is 20mV/s;

Figure 4 is the differential pulse voltammogram of 10 μmol/L DA in 0.1mol/L phosphate buffer solution on the ultrafine gold disk electrode at a scan rate of 20mV/s;

Figure 5 is a cyclic voltammogram of 1.0mmol/L potassium ferricyanide in 0.5mol/l potassium chloride solution on a carbon fiber/platinum ultramicro composite electrode and a carbon fiber disk electrode with a sweep rate of 100mV/s.

Its figure 1:

1. Ultramicro working electrode, 2. Glass capillary, 3. Conductive adhesive, 4. Conductor, 5. Insulation layer, 6. Metal layer, 7. Epoxy resin; M, end face, N, side.

【Detailed ways】

The invention provides a method for combining ultrafine combined electrodes with wide application and small volume, the ultrafine combined electrodes obtained by the method and the preparation process of the ultrafine combined electrodes.

First of all, the present invention provides a combination method of electrochemical ultrafine combined electrodes, which is to integrate the ultrafine working electrode and the alignment/quasi reference electrode to form a two-electrode system, the ultrafine working electrode and the alignment/alignment The reference electrodes are separated by a nanometer-thick insulating layer. In this way, the diffusion layers on the two electrodes can overlap, and the electrochemically reversible particles can be oxidized or reduced on one electrode, while on the other electrode they can be reduced or reduced. Oxidation is regenerated and then diffused back to the original electrode. Since the ultramicro working electrode and the alignment/quasi-reference electrode are integrated together, after the ultramicro-working electrode and the alignment/quasi-reference electrode are separated by an insulating layer, the two electrodes are in a short-circuit state at this time. According to the mass transfer and diffusion theory of the ultra-micro disk electrode, the expression of the mass transfer rate Ms is as follows:

${\mathrm{<span\; class="notranslate">\; m</span>}}_{\mathrm{<span\; class="notranslate">\; the\; s</span>}}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; 4</span>}\mathrm{<span\; class="notranslate">\; D.</span>}}{\mathrm{<span\; class="notranslate">\; \&pi;r</span>}}$

In the formula, D is the diffusion coefficient of the particle, and r is the radius of the disk electrode. In the time t, the moving distance of the particles in the diffusion field, that is, the thickness of the diffusion layer is M _s t. Assuming that r=3μM, the typical diffusion coefficient of particles in aqueous solution D=5×10 ^-6 cm ² /s, when the scan rate is kV/s, the time required to complete a scan is on the order of ms, so the thickness of the diffusion layer can reach several Hundreds of nm, with the increase of scanning speed, the thickness of diffusion layer becomes thinner correspondingly. Obviously, by properly controlling the thickness of the insulating layer, the diffusion layer on the ultramicro working electrode and the alignment/quasi-reference electrode will overlap, and at this time, the electrochemically reversible particles are oxidized or reduced on one electrode, while on the other electrode It is regenerated through reduction or oxidation, and then diffuses back to the original electrode, so a redox cycle is generated to amplify the output current signal, and the amplification factor depends on the thickness of the insulating layer and the dynamics and electrical properties of the particle. Chemical properties, generally speaking, the thinner the insulating layer, the faster the reaction kinetics of the particles, and the greater the magnification. The scan rates available for analytical studies have also been increased.

With the above theoretical basis, the structure of the ultramicro composite electrode in the present invention is interpreted according to FIG. 1 . As shown in Fig. 1, a kind of electrochemical ultramicro composite electrode designed according to the above-mentioned method of the present invention comprises ultramicro working electrode 1 and glass capillary 2, and described ultramicro working electrode 1 part is placed in glass capillary 2, The end point of the ultra-micro working electrode 1 inserted in the glass capillary 7 is connected to a wire 4.1 through the conductive glue 3, and the wire 4.1 is sealed and fixed in the glass capillary 2 with epoxy resin 7, and its end is exposed outside the glass capillary 2; The outer surface of the other end of the ultra-micro working electrode 1 is evenly coated or aggregated with an insulating layer 5 with a thickness of one layer of nanometer, and the insulating layer 5 and the outer surface of the glass capillary 7 are evenly coated with a metal layer 6. Layer 6 is led out from another wire 4.2 to become the above-mentioned alignment/quasi-reference electrode, forming an integrated combined electrode structure of the ultramicro working electrode and the alignment/quasi-reference electrode.

In the above combined electrode structure, the ultramicro working electrode 1 can be a carbon base electrode or a metal electrode, wherein the carbon base electrode can be made of carbon fiber material or carbon nanomaterial, and the metal electrode can be made of metal materials such as gold and platinum. The advantage of using the above materials is that the fabricated electrode has good biocompatibility, has electrochemical catalytic effect on some biomolecules, is convenient to purchase, and is simple to manufacture.

The insulating layer 5 is made of insulating varnish, epoxy resin or high molecular polymer, wherein the insulating varnish can be anodic electrophoretic paint, cathodic electrophoretic paint or automobile primer, etc., and the high molecular polymer can be poly-o-phenylenediamine, poly Electrochemically inert polymers such as vinylpyridine, poly(L-lysine), phenol and 2-allylphenol copolymers, the advantage of using the above materials is that the insulating layer 5 formed therefrom has a good insulating effect and a dense insulating film. Uniform, good stability.

Described metal layer 6 is made of precious metal materials such as gold, platinum, silver or rare metal material (as rhodium, iridium etc.), adopts the advantage of above-mentioned material to be that the alignment/quasi-reference electrode that constitutes thus has strong electrical conductivity, electrochemical properties Stable, the surface is easy to clean and not easily polluted.

The preparation process of the ultrafine composite electrode structure designed according to the above combination method is:

First, one end of the ultramicro working electrode 1 is bonded to the wire 4.1 through the conductive glue 3, and then a part of the electrode is sealed and fixed in the glass capillary 2, and the end of the wire 4.1 is exposed outside the glass capillary 2; The surface of the unsealed end of the glass capillary 2 is uniformly coated or aggregated with an insulating layer 5 with a thickness of nanometers, and then a metal layer 6 is evenly coated on the insulating layer 5 and the outer surface of the glass capillary 2, and is drawn out by a wire 4.2 to form a Align/align the reference electrode, and finally cut off the front end of the electrode coated with the metal layer 6 and the insulating layer 5 vertically, and the section of the exposed ultramicro working electrode is used as an ultramicro combined electrode.

In the above manufacturing process, the coating process of the insulating layer 5 is as follows: immerse the ultrafine working electrode 1 in insulating varnish or epoxy resin, take it out after 1 to 5 minutes, and then dry it at 60°C to 80°C. The thickness of the insulating layer 5 is controlled between 100-500nm. Experiments have proved that the selection of the insulating layer with the above thickness can overlap the diffusion layer of the electroactive particles on the working electrode and the alignment/quasi-reference electrode at a medium scan rate, and the electrochemical response signal can be regenerated and amplified, thereby improving the detection sensitivity.

The polymerization process of the insulating layer 5 is as follows: immerse the ultramicro working electrode 1 in a solution of phenol and 2-allylphenol, and then apply a voltage of 2V to 5V on the ultramicro working electrode 1 to generate the above two chemical substances Polymerization, a polymeric insulating layer can be formed on the surface of the ultramicro working electrode 1, and the thickness of the insulating layer 5 is controlled between 10-50nm. Experiments have proved that choosing an insulating layer with the above thickness can greatly reduce the solution resistance, greatly increase the available scan rate for actual system analysis, and facilitate the study of electron transfer and chemical reaction kinetics of the system.

The coating process of the metal layer 6 is: the evaporation or sputtering method (for the prior art process) coats the outer wall of the insulating layer 5 and the glass capillary 2 with precious metals or rare metals such as gold, platinum, silver, and the metal layer 6 The thickness is controlled between 50-80nm. Experiments have proved that the metal layer with the above-mentioned thickness has good conductivity, is stable, and is not easy to fall off.

In order to remove impurities on the electrode surface and further improve the detection sensitivity and detection quality, the ultramicro working electrode 1 is cleaned by a cleaning process before bonding the wire 4.1 and sealing it in the glass capillary 2, wherein the ultramicro working electrode 1 made of carbon material The working electrode 1 is ultrasonically cleaned with acetone, ethanol, double distilled water or a mixture thereof and dried. The ultrafine working electrode 1 made of gold or platinum metal is cleaned with nitric acid and aqua regia and dried.

The characteristics and advantages of the present invention will be analyzed through specific examples, applications and accompanying drawings of the preparation process.

Embodiment 1: carbon fiber/gold (CF/Au) ultrafine composite electrode

In this embodiment, carbon fiber electrodes and gold are selected as the combination of the two electrodes, and ethanol and double distilled water are used for ultrasonic cleaning; the conductive adhesive is silver conductive adhesive; the insulating layer is formed by polymerization. The specific production process is as follows: first place the carbon fiber electrode in acetone to reflux for 8 hours, then ultrasonically clean it with ethanol and double distilled water, cut off a 3cm length after complete drying, and then bond it to the end of a 0.1mm copper wire with silver conductive adhesive , placed in a glass capillary of 1.0mm, the carbon fiber electrode was sealed in the glass capillary on the glass capillary puller, and the copper wire exposed at the other end of the glass capillary was sealed and fixed with epoxy resin; then the carbon fiber electrode was immersed in phenol and In the 2-allylphenol solution, maintain a constant potential of 4V on the carbon fiber electrode for about 5-7min, so that phenol and 2-allylphenol can be polymerized on the surface of the carbon fiber, and after baking at 140°C, a layer with a thickness of about 10nm is formed. For the insulating layer, a layer of gold with a thickness of about 70nm is evaporated and plated on the insulating layer, and it is drawn out with a copper wire as an alignment/quasi-reference counter electrode. Finally, the front end of the carbon fiber electrode coated with the gold layer and the insulating layer is cut off vertically. That is, a carbon fiber/gold (CF/Au) ultramicro composite electrode is obtained. Using the same treatment method as above, after the insulating layer is formed on the surface of the carbon fiber electrode, the front end of the electrode is cut off directly, without the gold-plated layer, and the carbon fiber disk electrode is obtained.

The carbon fiber/gold (CF/Au) ultrafine composite electrode and carbon fiber disc surface electrode prepared above were used to detect anthracene for comparison: respectively insert 12mmol/L anthracene acetonitrile solution with 0.1mol/L NEt ₄ BF ₄ as supporting electrolyte Among them, the cyclic voltammetry characteristics were measured at a scan rate of 1.34MV/s, and Figure 2 was obtained. The volt-ampere curve a in the figure uses the traditional two-electrode system with a carbon fiber disk electrode as the working electrode. Due to the low concentration of the supporting electrolyte and the large R _s , 100% compensation can no longer be achieved under the premise of maintaining the circuit bandwidth, and only about 40% can be compensated. %, the Faraday peak cannot be resolved; the curve b uses ultra-micro-combined electrodes, and the Faraday peak can be clearly seen because the R _s is significantly reduced and the ohmic drop is reduced, but if it is not compensated, the ohmic drop still remains, so the peak spacing ΔE _p larger than the theoretical value. But at this time, the ohmic drop can be fully compensated by the electronic positive feedback technology, as shown by the volt-ampere curve c. At this time, the experimental curve c basically coincides with the simulation curve d.

Embodiment 2: gold/platinum (Au/Pt) ultramicro combined electrode

The preparation process of gold/platinum (Au/Pt) ultrafine composite electrode is similar to that of Example 1. After confirming the various materials and methods used, at first the gold wire with a diameter of 5nm is cleaned in nitric acid and double distilled water, dried and used The conductive adhesive is bonded to the copper wire, placed in a capillary to draw and seal, and the surface of the gold wire is coated with insulating varnish. After drying, an insulating layer with a thickness of 120nm is obtained, and then a layer of 50nm thick platinum is sputtered on the outside of the insulating layer. , using a copper wire to lead out as an alignment/quasi-reference counter electrode, and finally, the front end of the gold electrode coated with a platinum layer and an insulating layer is cut vertically to obtain a gold/platinum (Au/Pt) ultramicro composite electrode. Using the same treatment method as above, after the insulating layer is formed on the surface of the gold electrode, the front end of the electrode is cut off directly, and the platinum layer is no longer sputtered, that is, the ultrafine gold disk electrode is obtained.

The gold/platinum (Au/Pt) ultramicro composite electrode and ultrafine gold disk electrode prepared above are used to detect the neurotransmitter dopamine for comparison: insert in 0.1mol/L phosphate buffer solution of 10 μmol/L DA, at 20mV Measure the differential pulse voltammetry characteristics at 20mV/s to obtain Figure 3; insert the ultrafine gold disk electrode into the same solution as above, and measure the differential pulse voltammetry characteristics at 20mV/s to obtain Figure 4. Comparing the curve in Figure 3 with the curve in Figure 4, it can be seen that the differential pulse peak current of the electrochemical oxidation of DA on the Au/Pt ultrafine composite electrode is nearly 10,000 times higher than that on the ultrafine gold disk electrode.

Embodiment 3: carbon fiber/platinum (CF/Pt) ultramicro combined electrode;

The cleaning treatment of the carbon fiber is the same as that of Example 1. A layer of epoxy resin is uniformly coated on the clean carbon fiber surface, and the thickness of about 450nm is about 450nm after complete drying. A layer of 50nm thick platinum is sputtered on the epoxy surface, and a copper wire is used to draw it as To align/quasi-reference the counter electrode, finally, the front end of the carbon fiber electrode coated with the platinum layer and the epoxy insulating layer is cut vertically to obtain a carbon fiber/platinum (CF/Pt) ultramicro composite electrode. Using the same treatment method as above, after the insulating layer is formed on the surface of the carbon fiber electrode, the front end of the electrode is cut off directly, and the platinum layer is no longer sputtered, that is, the carbon fiber disk electrode is obtained.

The carbon fiber/platinum (CF/Pt) ultramicro combined electrode and the carbon fiber disc surface electrode prepared above are used to detect potassium ferricyanide for comparison: the CF/Pt ultrafine combined electrode is inserted into 0.5 of 1.0mmol/L potassium ferricyanide In the mol/l potassium chloride solution, measure the cyclic voltammetric characteristic at 100mV/s, and obtain the curve a in the accompanying drawing 5; insert the carbon fiber disk electrode into the above-mentioned same solution, measure the cyclic voltammetric characteristic at 100mV/s , to obtain curve b in accompanying drawing 5. Comparing the curve a with the curve b, it can be seen that the electrochemical response of potassium ferricyanide on the CF/Pt ultramicro composite electrode is nearly 4 times higher than that on the carbon fiber disk electrode.

Through the examples of the above three preparation processes and the specific comparative analysis, it can be seen that the ultrafine combined working electrode prepared according to the combination method of the present invention overcomes the large solution resistance and low scan rate of the conventional electrode system in the detection. , large interference to the research system, etc., can significantly reduce the solution resistance, and the electrochemically reversible particles will undergo a redox regeneration cycle on the combined electrode to amplify the output current signal, so the actual system analysis can be greatly improved. The scanning speed and detection sensitivity minimize the damage to the research system. The ultra-micro-combined electrode is easy to manufacture, has a long life and is easy to update. It is a high-performance electrochemical biosensor that can be used for high spatiotemporal resolution of single cell release. Dynamic detection and real-time dynamic analysis of single vesicles in cells.

Claims (10)

1, a kind of combined method of galvanochemistry ultramicroelectrode, it is characterized in that: it is that ultra micro working electrode and right/accurate contrast electrode combination are integrated, form two electrode systems, insulation course by nano thickness between described ultra micro working electrode and the right/accurate contrast electrode is isolated, make particle oxidation or reduction on an electrode of the diffusion layer generation coincidence back electrochemical reversible on two electrodes, and on another electrode, pass through to reduce or oxidation regeneration, and then return and diffuse on the original electrode, thereby the current signal of exporting on the electrode of surveying cell is exaggerated.

2, a kind of electrochemical ultra-micro compound electrode is characterized in that: it comprises ultra micro working electrode, right/accurate contrast electrode and glass capillary, and described ultra micro working electrode one end seals and is fixed in the glass capillary, and its end points is connected with lead by conducting resinl; Even coating in described ultra micro working electrode other end surface or polymerization have the insulation course of a nano thickness, and this insulation course and described glass capillary outside surface evenly are coated with a metal level, and this metal level is drawn by another lead, constitutes right/accurate contrast electrode.

3, electrochemical ultra-micro compound electrode according to claim 2 is characterized in that: described ultra micro working electrode is carbon material basic electrode or metal ultramicroelectrode.

4, electrochemical ultra-micro compound electrode according to claim 2 is characterized in that: described insulation course is made of insullac, epoxy resin or high molecular polymer.

5, electrochemical ultra-micro compound electrode according to claim 2 is characterized in that: described right/accurate contrast electrode is made of gold, platinum, silver-colored precious metal material or Rare Metals Materials.

6, electrochemical ultra-micro compound electrode according to claim 3 is characterized in that: described carbon material basic electrode material is carbon fiber or carbon nanomaterial, and described metal ultramicroelectrode material is gold or platinum.

7, a kind of preparation technology of electrochemical ultra-micro compound electrode is characterized in that comprising following concrete steps:

Earlier that ultra micro working electrode one end is bonding by conducting resinl and lead, then this end is sealingly fastened in the kapillary, lead is exposed to extracapillary; Evenly apply in the unencapsulated surface of the ultra micro working electrode other end again or the insulation course of polymerization one nano thickness, evenly plate a metal level at this insulation course and glass capillary outside surface then, with lead draw make its formation right/accurate contrast electrode, to be coated with vertical cut-out of electrode front end of metal level and insulation course at last, the ultra micro working electrode cross section of exposing is as ultra-micro compound electrode.

8, the preparation technology of electrochemical ultra-micro compound electrode according to claim 7, it is characterized in that: described ultra micro working electrode is at the bonding lead and before being sealed in kapillary, the ultra micro working electrode that is made of the carbon material adopts acetone, ethanol, distilled water or its potpourri ultrasonic cleaning clean also dry, and the ultra micro working electrode that is made of gold or platinum metal material adopts nitric acid, chloroazotic acid to clean and drying.

9, the preparation technology of electrochemical ultra-micro compound electrode according to claim 7, it is characterized in that: the coating processes of described insulation course is for immersing the ultra micro working electrode in insullac or the epoxy resin, took out oven dry after 1～5 minute, the THICKNESS CONTROL of its insulation course is between 100-500nm; Being polymerized to of described insulation course immersed the ultra micro working electrode in phenol and the 2-allyl benzene phenol solution, on the ultra micro working electrode, apply the voltage of 2V～5V, make above-mentioned two kinds of chemical substance polymerizations, form the high molecular polymerization insulation course at the ultra micro working electrode surface, the THICKNESS CONTROL of its insulation course is between 10-50nm.

10, the preparation technology of electrochemical ultra-micro compound electrode according to claim 7, it is characterized in that: described metal level coating adopts the method for evaporation or sputter to plate one deck gold, platinum, silver metal or rare metal at insulation course and kapillary outer wall, and its metal layer thickness is controlled between the 50-80nm.

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