CN105842312B - Nanometer flower-shaped ultramicro gold electrode and preparation and application thereof - Google Patents
Nanometer flower-shaped ultramicro gold electrode and preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of preparation methods and application of microelectrodes, and provides a nanometer flower-shaped ultramicro gold electrode and a preparation method and application thereof. Adhering micron-sized carbon fibers and copper wires by using a graphite conductive adhesive, stretching into a glass capillary with a stretched end part, carrying out flame etching to reach a nanometer-sized diameter, carrying out electrochemical deposition of electrophoretic paint on the surface of the glass capillary, heating and baking, and carrying out electrochemical deposition of gold in an acid chloroauric acid solution to obtain the nanometer flower-shaped ultramicro gold electrode. The prepared electrode surface is assembled with biomolecules marked with electrochemical active substances, and can specifically recognize target molecule surface receptors and detect the target molecules by an electrochemical method. The electrode can be applied to the research fields of biosensors, biomolecule detection and the like. The preparation method is simple, low in cost, small in size and easy to operate; the prepared gold electrode has clean surface, increases the specific surface area of the ultramicroelectrode and enhances the electrochemical signal.
Description
Technical Field
The invention belongs to the field of preparation methods and application of microelectrodes, and relates to a preparation method of an ultramicro gold electrode, which can be applied to the research fields of biosensors, biomolecule detection and the like.
Background
The microelectrode is one of important branches of modern electrochemistry disciplines, and has high superiority in trace detection, single cell detection and biosensing. The ultramicroelectrode has extremely small size, the diameter can reach micron-scale or even nanometer-scale, and the ultramicroelectrode is an advantageous means for exploring the characteristics of microscopic substances and can also be used for detecting neurotransmitter information in real time. In addition, the ultramicroelectrode has high current density, low RC constant and fast mass transfer rate, and the excellent characteristics enable the ultramicroelectrode to have fast response speed and high signal-to-noise ratio. Based on the above advantages, scientific researchers have been working on developing more convenient and efficient methods for preparing ultramicroelectrodes.
In the field of electrochemical research, gold electrodes are one of the most commonly used electrodes in electrochemical research and application due to their excellent electrochemical properties and easy modification. With the progress of electrochemical research, researchers have been working on developing various methods for preparing gold ultramicroelectrodes in recent years. In the present study, the following methods are mainly used for preparing the gold ultramicroelectrode: firstly, gold nano-electrodes (adv. Mater.,2010,22, 2148-; or the gold ultramicroelectrodes with controllable size can be prepared after the gold is catalyzed and reduced at the tip of the drawn quartz and then encapsulated by electrophoretic paint (biosens, bioelectrectro, 2015, 262-268). However, in both methods, the gold surface is encapsulated with electrophoretic paint at a later stage in order to obtain a smaller gold area, which may cause impurities on the gold surface and may affect later research. And secondly, gold is reduced and deposited on the carbon fiber nanometer disk encapsulated by the plastic packaging film to prepare a gold ultramicroelectrode (Yangzhou university journal (Nature science edition), 2006,9, 21-25). The disadvantage of this method is that it requires a high grinding process and produces electrodes with a large volume and a small effective area (about tens to hundreds of nanometers). And thirdly, depositing gold on a photoetching silicon wafer to obtain a nano golden flower microelectrode (nat. chem.,2012,4, 642-648). However, such a micro-electrode may have a limitation in practical use due to a large volume of the substrate. And fourthly, depositing gold after chemically etching the mechanically drawn platinum nano electrode to prepare a nano golden flower electrode (anal. chem.,2014,86,2849-2852), but the method has higher requirements on instrument equipment and cost. Due to the limitations of the preparation methods, the popularization and the application of the gold ultramicroelectrode in various fields are hindered to a certain extent. Therefore, it is very important to provide a method for preparing gold ultramicroelectrodes, which is simple, low in cost and easy to operate.
The invention provides a preparation method of a nanometer flower-shaped ultramicro gold electrode, which overcomes the technical problems, does not need complex and expensive instruments and equipment and a grinding process which is difficult to operate, and has the advantages of extremely low impurity content, low manufacturing cost and large proportion of effective area. The gold electrode prepared by the method can be used in the research fields of biosensors, biomolecule detection and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of a nanometer flower-shaped ultramicro gold electrode. The gold electrode is obtained by electrophoretic paint deposition and gold deposition on the basis of carbon fibers. The specific technical scheme of the preparation method is as follows.
A preparation method of a nanometer flower-shaped ultramicro gold electrode comprises the following steps:
the method comprises the following steps: adhering the copper wire and the micron-sized carbon fibers by using a graphite conductive adhesive;
step two: axially extending the copper wire adhered with the carbon fiber into the glass capillary with the stretched end part;
step three: the tail part of the capillary tube is sealed by epoxy resin;
step four: the tip of the capillary tube is sealed by flame fusion, and the carbon fiber is exposed out of the tip of the glass capillary tube for flame etching;
step five: placing the end part of the etched carbon nanofiber into an electrophoretic paint solution for electrochemical deposition;
step six: heating and baking the electrode on which the electrophoretic paint is deposited;
step seven: and (3) carrying out electrochemical deposition on the treated electrode in an acid chloroauric acid solution to obtain the nano flower-shaped ultramicro gold electrode with the end part of the flower-shaped gold nanostructure.
And C, cutting the carbon fiber in the step I into short sections with the diameter of 7 micrometers and the length of 1.5-2 cm, respectively ultrasonically cleaning the short sections for 3min by using acetone, ethanol and ultrapure water, cleaning the short sections for 3-5 times by using the ultrapure water, and drying the short sections in a 37 ℃ thermostat. The length of the copper wire is 8-10 cm.
The diameter of the glass capillary tube in the second step is 0.8-1.5 mm, the diameter of the copper wire is smaller than the inner diameter of the glass capillary tube, and the inner diameter of the tip of the drawn glass tube is about 50-100 mu m.
And the flame in the fourth step is the flame of an alcohol lamp, and the tip of the capillary tube is sealed by flame fusing.
The diameter of the carbon fiber after etching in the fourth step is 100-500nm, and the length is 100-300 μm.
And in the fifth step, the electrophoretic paint is cathode electrophoretic paint, and the electrophoretic paint solution is obtained by mixing the electrophoretic paint and ultrapure water in a certain proportion and ultrasonically treating the mixture.
And in the fifth step, the electrochemical deposition is carried out in a double-electrode system, carbon fibers are used as working electrodes, platinum wires are used as counter electrodes, the deposition voltage is-8V, and the deposition time is 100 s.
And the heating and baking step in the sixth step is finished in an oven, the temperature is 60-80 ℃, and the baking time is 5-10 min.
The seven acidic chloroauric acid solutions in the step are obtained by mixing hydrochloric acid and chloroauric acid, and the concentration of the hydrochloric acid and the chloroauric acid solutions is 0.5mol/LHCl and 20mmol/L HAuCl4。
And seventhly, performing electrochemical deposition in a three-electrode system, wherein carbon fibers are used as a working electrode, a platinum wire is used as a counter electrode, Ag/AgCl (3M KCl) is used as a reference electrode, the deposition voltage is 0V, and the deposition time is 300 s.
The preparation method comprises the steps of adhering micron-sized carbon fibers and copper wires by using a graphite conductive adhesive, extending the carbon fibers and the copper wires into a glass capillary with a stretched end part, carrying out flame etching to reach a nanometer-sized diameter, carrying out electrochemical deposition of electrophoretic paint on the surface of the glass capillary, heating and baking the glass capillary, and carrying out electrochemical deposition of gold in an acidic chloroauric acid solution to obtain the nanometer flower-shaped ultramicro gold electrode. The method comprises the steps of firstly depositing the electrophoretic paint and then depositing the gold, wherein the prepared gold electrode almost does not contain impurities and does not influence later-stage research and practicability; the grinding process which is not easy to operate is not adopted, but a flame etching technology which is easy to control is adopted, so that the prepared gold electrode is small in size, and the proportion of the effective area is large; the stretched capillary tip is used as a bearing substrate, the substrate is small in size and can be used in various use environments; the preparation process does not use large-scale complicated instruments, and the preparation cost is reduced. Generally, the preparation method provided by the invention can effectively prepare the ultramicro gold electrode, improve the preparation success rate of the ultramicro gold electrode, is simple to operate and reduces the preparation cost.
The invention also provides a nano flower-shaped ultramicro gold electrode prepared by the method, which consists of copper wires, glass capillaries, epoxy resin, carbon fibers, graphite conductive adhesive, electrophoretic paint and flower-shaped gold nano structures. The end part of the gold electrode is of a flower-shaped structure, the gold is used as a component, and the diameter of the flower-shaped structure is 50-300 mu m.
After the copper wire is adhered to the carbon fiber, the copper wire axially penetrates into the drawn glass capillary tube, and the tail end of the copper wire is sealed by epoxy resin; and melting the tip of the heated glass capillary tube to seal the carbon fiber, etching the carbon fiber to a nano level by using flame, sealing the carbon fiber by using electrophoretic paint, curing and shrinking the baked electrophoretic paint to expose carbon fiber with a certain area, and forming a flower-shaped gold nanostructure at the end by performing electrochemical deposition in acid chloroauric acid, thereby preparing the nano flower-shaped ultramicro gold electrode. Golden flowers do not grow on the part of the carbon fiber encapsulated by the electrophoretic paint, and flower-shaped structures grow on the exposed tip parts of the carbon fiber after baking. The size of the flower-like structure is in the nanometer level as seen by scanning electron microscopy.
In the preparation method, the length and the diameter of the carbon fiber after flame etching, the electrophoretic paint deposition process condition, the gold deposition process condition and the like are factors influencing the diameter of the flower-shaped structure, and the ultramicro gold electrode with the flower-shaped structures with different diameters can be prepared by changing the factors.
The invention also provides an application of the nano flower-shaped ultramicro gold electrode in electrochemical biosensing. The prepared gold electrode surface is assembled with biomolecules marked with electrochemical active substances, the biomolecules are identified with target molecules to cause the change of the distance between the electrochemical active molecules and the gold surface, and the electrochemical active molecules are detected by an electrochemical workstation, so that the electrochemical detection of the target molecules can be specifically realized. The application process is as follows:
(1) assembling the biomolecule marked with electrochemical active substances and the nano flower-shaped ultramicro gold electrode: assembling biomolecules with the surface of a gold electrode through sulfydryl modification, wherein the marked electrochemical active substances are micromolecules with electrochemical activity such as methylene blue, ferrocene and the like, the biomolecules are polypeptide and DNA (aptamer), and the assembling condition is that the biomolecules are assembled overnight at room temperature in a salt solution with certain concentration in a dark place;
(2) and MCH is sealed: placing the assembled electrode in a 2mmol/L Mercaptohexanol (MCH) solution for reacting for 2 hours at room temperature so as to regulate and control the assembly density of the surface probe;
(3) detection of target molecules: and (3) placing the gold electrode prepared in the step (2) in an electrochemical detection solution, and measuring electrochemical signals of the electrochemical active substances before and after the addition of the target molecules by adopting a three-electrode system through Alternating Current Voltammetry (ACV) or Square Wave Voltammetry (SWV).
The target molecule is one of cell, protein, and small molecule (such as cocaine and dopamine).
The flower-shaped nano structure of the gold ultramicroelectrode prepared by the method greatly increases the specific surface area of the ultramicroelectrode, and has important significance for enhancing electrochemical signals. The prepared electrode surface is assembled with biomolecules marked with electrochemical active substances, can specifically recognize target molecule surface receptors, and can detect the target molecules by an electrochemical method.
The invention has the following beneficial effects: 1. carrying out gold deposition after the electrophoretic paint is encapsulated, and cleaning the surface of the obtained gold electrode; 2. the preparation method is simple, low in cost, small in size and easy to operate; 3. the specific surface area of the ultramicroelectrode is increased, and the electrochemical signal is enhanced; 4. can be further assembled with biological molecules and is applied to the research fields of biosensors, biological molecule detection and the like.
Drawings
The invention will be further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of the preparation of a nano-gold-patterned electrode according to the present invention;
FIG. 2 is a schematic structural diagram of a nanoflower-shaped ultramicro gold electrode according to the present invention;
FIG. 3 is a scanning electron microscope photograph of the nano-flower-like ultrafine gold electrode of example 1 of the present invention;
FIG. 4 is a scanning electron microscope image of the surface topography of the flower-like structure of the ultramicro gold electrode of example 1 of the present invention;
FIG. 5 is a steady state voltammogram of a carbon fiber electrode before and after electrodeposition of an electrodeposition paint in example 1 of the present invention;
FIG. 6 is a cyclic voltammogram of the nano-flower-like ultramicro gold electrode of example 1 of the present invention;
FIG. 7 shows the percentage of enhancement of electrochemical signals of cocaine with different concentrations in the detection system measured by Alternating Current Voltammetry (ACV) after the cocaine aptamer labeled with methylene blue is assembled on the nano-flower-shaped ultramicro gold electrode in example 4 of the present invention;
fig. 8 shows that the percentage of the electrochemical signal enhancement of dopamine with different concentrations in the detection system is measured by Alternating Current Voltammetry (ACV) after the dopamine aptamer labeled with methylene blue is assembled on the nano flower-shaped ultramicro gold electrode in example 5 of the present invention.
Wherein, 1 is copper wire, 2 is glass capillary, 3 is epoxy, 4 is the carbon fiber, 5 is graphite conducting resin, 6 is the electrophoresis lacquer, 7 is flower gold nanometer structure.
Detailed Description
The present invention will be described in further detail below with reference to the accompanying drawings and examples. The following example is one example of the technical solution of the present invention, and the protection scope of the present invention is not considered to be limited to the following example.
Example 1:
FIG. 1 is a schematic diagram of the preparation of a nano-gold-patterned electrode according to the present invention. The preparation process of the nano flower-shaped ultramicro gold electrode comprises the following steps:
the method comprises the following steps: cutting 7 mu m carbon fiber 4 into short sections with the length of 1.5 cm-2 cm, carrying out ultrasonic cleaning for 3min by using acetone, ethanol and ultrapure water in sequence, then cleaning for 3 times by using the ultrapure water, and drying in a 37 ℃ thermostat. And cutting a copper wire 1 with the length of about 10cm, and adhering the copper wire 1 with the carbon fiber 4 after cleaning and drying by using a graphite conductive adhesive 5.
Step two: one end of a glass capillary 2 having a diameter of 0.8mm was drawn to an inner diameter of about 50 μm at the tip, and a copper wire 1 having carbon fibers 4 adhered thereto was inserted from the thicker end of the glass capillary 2 to expose about 0.5mm at the tip.
Step three: the tail of the glass capillary 2 is closed with an epoxy resin 3.
Step four: after the tip of the capillary 2 was melted and sealed with an alcohol lamp, the carbon fiber 4 exposed outside the tip of the glass capillary was continuously flame-etched on the alcohol lamp to a length of about 200 μm and a tip diameter of about 500 nm. And ultrasonically cleaning the prepared carbon fiber electrode at low frequency.
Step five: the tip of the electrode was immersed in an electrophoretic paint solution (the mass ratio of the electrophoretic paint to ultrapure water was 1:5, and subjected to ultrasonic treatment) at a ratio of 1:5 for electrochemical deposition, and the immersion depth was about 1cm, and in order to allow the electrophoretic paint 6 to be uniformly attached to the surface of the carbon fiber 4, the platinum wire was bent into a ring shape (a diameter of about 1.5 cm), and the electrode immersion position was located at the center of the platinum wire ring. The carbon fiber ultramicroelectrode is used as a working electrode, a platinum wire is used as a counter electrode, an electrochemical workstation 'Amperometric i-t Curve' mode of CHI760E is selected, the deposition voltage is-8V, and the deposition time is 100 s. After the deposition, the electrode was taken out, and the residue remaining on the surface of the electrode tip was rinsed off with ultrapure water.
Step six: and vertically and upwards placing the electrode subjected to electrophoretic paint deposition in an oven at 80 ℃ for baking for 5min, so that the electrophoretic paint is cured and shrunk, and the surface of the carbon fiber with a certain size is exposed. At 10mmol/L K3[Fe(CN)6]The steady voltammetric current of the detection electrode in the solution (containing 0.5mol/L KCl) was 10mV/s in sweep rate, and the results are shown in FIG. 5. Regarding the carbon fiber electrode after encapsulation as a hemispherical electrode approximately, using a formula: i.e. i0＝2πnFDcr0The effective radius of the electrode is calculated, and the calculation result is about 250 nm.
Step seven: screening carbon fiber electrodes with similar effective radius, and soaking the carbon fiber electrodes in an acidic chloroauric acid solution, wherein the chloroauric acid solution is prepared by HAuCl with the concentration of 20mmol/L4And 0.5mol/L HCl, and performing electrodeposition on the electrode by adopting a three-electrode system, wherein the carbon fiber 4 is a working electrode, the platinum wire is a counter electrode, and Ag/AgCl (3M KCl) is a reference electrode. CHI760E electrochemical workstation "Amperometric i-t Curve" mode was chosen, deposition voltage 0V and deposition time 300 s. After the deposition is completed, the electrode is taken out and carefully cleaned by ultrapure water, and finally the nano flower-shaped ultramicro gold electrode with the end part of the flower-shaped gold nano structure 7 is prepared.
Fig. 2 is a schematic structural view of the nano flower-shaped ultramicro gold electrode of the present invention, which is prepared by the above preparation method, and as shown in fig. 2, the nano flower-shaped ultramicro gold electrode of the present invention is composed of a copper wire 1, a glass capillary 2, an epoxy resin 3, a carbon fiber 4, a graphite conductive adhesive 5, an electrophoretic paint 6, and a flower-shaped gold nanostructure 7. After the copper wire 1 is adhered to the carbon fiber 4, the copper wire axially penetrates into the drawn glass capillary tube 2, and the tail end of the copper wire is sealed by epoxy resin 3; the tip of the heated glass capillary tube 2 is melted to seal the carbon fiber 2, the carbon fiber 2 is etched to be nanoscale by flame, then is encapsulated by the electrophoretic paint 6, the baked electrophoretic paint 6 is cured and contracted to expose carbon fiber with a certain area, and electrochemical deposition is carried out in acid chloroauric acid to form a flower-shaped gold nano structure 7 at the end part, so that the nanometer flower-shaped ultramicro gold electrode is prepared.
Fig. 3 is a scanning electron microscope image of the nano-flower-like ultrafine gold electrode of the present example. It can be seen that no golden flower grows on the part of the carbon fiber encapsulated by the electrophoretic paint, and the tip part of the exposed carbon fiber grows into a flower-shaped structure after baking. The tip portion of the electrode is flower-like in structure and has a diameter of about 100 μm.
FIG. 4 is a scanning electron microscope image of the surface topography of the flower-like structure of the ultramicro gold electrode in this embodiment. The size of the surface flower-like structures can be seen to be in the nanometer scale.
FIG. 5 shows the carbon fiber electrode at 10mmol/L K before and after electrophoretic paint deposition in this example3[Fe(CN)6]Steady state voltammograms in solution (containing 0.5mol/LKCl) at a scan rate of 10 mV/s. The carbon fiber electrodes before and after encapsulation in FIG. 5 can obtain a typical "S" -shaped steady-state voltammogram of the ultramicroelectrode. In fig. 5, a gray solid line shows the carbon fiber ultramicroelectrode after electrophoretic paint deposition, and compared with a steady voltammetry curve (a black solid line) before deposition, the steady voltammetry current value of the encapsulated ultramicroelectrode is obviously reduced, which indicates that the effective radius of the electrode is reduced.
FIG. 6 is a cyclic voltammogram of the nanoflower-shaped ultramicro gold electrode of this example in 0.05mol/L sulfuric acid (4 scanning fragments). It can be seen that a characteristic reduction peak of gold in sulfuric acid occurs at 0.863V, indicating that gold is deposited at the electrode tip. In addition, the obtained characteristic reduction peak area is large and is equivalent to the reduction peak area of a common disk electrode (with the diameter of about 2mm) in sulfuric acid, and a scanning electron microscope image shows that the size of the deposited golden flower is only 100 μm, which indicates that the electrode has a large specific surface area.
Example 2:
the preparation process of the nano flower-shaped ultramicro gold electrode comprises the following steps:
the method comprises the following steps: cutting 7 mu m carbon fiber 4 into short sections with the length of 1.5 cm-2 cm, carrying out ultrasonic cleaning for 3min by using acetone, ethanol and ultrapure water in sequence, then cleaning for 5 times by using the ultrapure water, and drying in a 37 ℃ thermostat. And cutting a copper wire 1 with the length of about 8cm, and adhering the copper wire 1 with the carbon fiber 4 after cleaning and drying by using a graphite conductive adhesive 5.
Step two: one end of a glass capillary 2 having a diameter of 1.5mm was drawn to an inner diameter of about 100 μm at the tip, and a copper wire 1 having carbon fibers 4 adhered thereto was inserted from the thicker end of the glass capillary 2 to expose about 0.5mm at the tip.
Step three: the tail of the glass capillary 2 is closed with an epoxy resin 3.
Step four: after the tip of the capillary 2 was melted and sealed with an alcohol lamp, the carbon fiber 4 exposed outside the tip of the glass capillary was continuously flame-etched on the alcohol lamp until the length was about 100 μm and the tip diameter was about 100 nm. And ultrasonically cleaning the prepared carbon fiber electrode at low frequency.
Step five: the tip of the electrode was immersed in an electrophoretic paint solution (the mass ratio of the electrophoretic paint to ultrapure water was 1:5, and subjected to ultrasonic treatment) at a ratio of 1:5 for electrochemical deposition, and the immersion depth was about 1cm, and in order to allow the electrophoretic paint 6 to be uniformly attached to the surface of the carbon fiber 4, the platinum wire was bent into a ring shape (a diameter of about 1.5 cm), and the electrode immersion position was located at the center of the platinum wire ring. The carbon fiber ultramicroelectrode is used as a working electrode, and the platinum wire is used as a counter electrode. CHI760E electrochemical workstation "Amperometric i-t Curve" mode was chosen, deposition voltage was-8V and deposition time was 100 s. After the deposition was completed, the electrode was taken out, and the residue remaining on the surface of the electrode tip was rinsed off with ultrapure water.
Step six: and vertically and upwards placing the electrode subjected to electrophoretic paint deposition in a 60 ℃ drying oven for baking for 10min, so that the electrophoretic paint is cured and shrunk, and the surface of the carbon fiber with a certain size is exposed.
Step seven: screening carbon fiber electrodes with similar effective radius, and soaking the carbon fiber electrodes in an acidic chloroauric acid solution, wherein the chloroauric acid solution is prepared by HAuCl with the concentration of 20mmol/L4And 0.5mol/L HCl, and performing electrodeposition on the electrode by adopting a three-electrode system, wherein the carbon fiber 4 is a working electrode, the platinum wire is a counter electrode, and Ag/AgCl (3M KCl) is a reference electrode. CHI760E electrochemical workstation "Amperometric i-t Curve" mode was chosen, deposition voltage 0V and deposition time 300 s. After the deposition is completed, the electrode is taken out and carefully cleaned by ultrapure water, and finally the nano flower-shaped ultramicro gold electrode with the end part of the flower-shaped gold nano structure 7 is prepared.
Example 3:
the preparation process of the nano flower-shaped ultramicro gold electrode comprises the following steps:
the method comprises the following steps: cutting 7 mu m carbon fiber 4 into short sections with the length of 1.5 cm-2 cm, carrying out ultrasonic cleaning for 3min by using acetone, ethanol and ultrapure water in sequence, then cleaning for 3 times by using the ultrapure water, and drying in a 37 ℃ thermostat. And cutting a copper wire 1 with the length of about 10cm, and adhering the copper wire 1 with the carbon fiber 4 after cleaning and drying by using a graphite conductive adhesive 5.
Step two: one end of a glass capillary 2 having a diameter of 0.8mm was drawn to an inner diameter of about 50 μm at the tip, and a copper wire 1 having carbon fibers 4 adhered thereto was inserted from the thicker end of the glass capillary 2 to expose about 0.5mm at the tip.
Step three: the tail of the glass capillary 2 is closed with an epoxy resin 3.
Step four: after the tip of the capillary 2 was melted and sealed with an alcohol lamp, the carbon fiber 4 exposed outside the tip of the glass capillary was continuously flame-etched on the alcohol lamp to a length of about 300 μm and a tip diameter of about 500 nm. And ultrasonically cleaning the prepared carbon fiber electrode at low frequency.
Step five: the tip of the electrode was immersed in an electrophoretic paint solution (the mass ratio of the electrophoretic paint to ultrapure water was 1:5, and subjected to ultrasonic treatment) at a ratio of 1:5 for electrochemical deposition, and the immersion depth was about 1cm, and in order to allow the electrophoretic paint 6 to be uniformly attached to the surface of the carbon fiber 4, the platinum wire was bent into a ring shape (a diameter of about 1.5 cm), and the electrode immersion position was located at the center of the platinum wire ring. The carbon fiber ultramicroelectrode is used as a working electrode, and the platinum wire is used as a counter electrode. CHI760E electrochemical workstation "Amperometric i-t Curve" mode was chosen, deposition voltage was-8V and deposition time was 100 s. After 100s reaction, the electrode was taken out, and the residue remaining on the surface of the electrode tip was rinsed off with ultrapure water.
Step six: and vertically and upwards placing the electrode subjected to electrophoretic paint deposition in an oven at 80 ℃ for baking for 5min, so that the electrophoretic paint is cured and shrunk, and the surface of the carbon fiber with a certain size is exposed.
Step seven: screening carbon fiber electrodes with similar effective radius, and soaking the carbon fiber electrodes in an acidic chloroauric acid solution, wherein the chloroauric acid solution is prepared by HAuCl with the concentration of 20mmol/L4And 0.5mol/L HCl, and performing electrodeposition on the electrode by adopting a three-electrode system, wherein the carbon fiber 4 is a working electrode, the platinum wire is a counter electrode, and Ag/AgCl (3M KCl) is a reference electrode. Selecting CHI760E electrochemical workstation "Amperometric i-t Curve" mode, deposition voltage is 0V,the deposition time was 300 s. After the deposition is completed, the electrode is taken out and carefully cleaned by ultrapure water, and finally the nano flower-shaped ultramicro gold electrode with the end part of the flower-shaped gold nano structure 7 is prepared.
Example 4:
the ultramicro gold electrode prepared in the embodiment 1 can be used in the field of electrochemical biosensing, and the specific process for sensing the aptamer comprises the following steps:
(1) assembling the biomolecule marked with electrochemical active substances and the nano flower-shaped ultramicro gold electrode: the ultramicro gold electrode prepared in example 1 was placed in an assembly solution of 100nmol/L cocaine aptamer labeled with thiol (SH) groups and Methylene Blue (MB), the assembly solution containing 1mol/L NaCl, 20mmol/L MgCl2And 20mmol/L PB assembled overnight at room temperature protected from light. Wherein, the sequence of the cocaine aptamer is as follows: 5' -SH- (CH)2)6GACAAGGAAAATCCTTCAATGAAGTGGGTC-MB-3」。
(2) And MCH is sealed: after assembly, the electrode is placed in 2mmol/L MCH solution for reaction for 2h, and the assembly density of the surface probe is regulated and controlled.
(3) Detection of target molecules: and (3) placing the gold electrode prepared in the step (2) in an electrochemical detection solution, performing electrochemical detection in an electrochemical workstation CHI760E, and using a three-electrode system, wherein the working electrode is a nano flower-shaped ultramicro gold electrode, the reference electrode is an Ag/AgCl (3M KCl) electrode, and the counter electrode is a platinum wire electrode. The measurement was carried out by Alternating Current Voltammetry (ACV). The electrochemical workstation "AC voltametry" mode was chosen to detect the electrochemical signal of methylene blue. since-0.28V is the characteristic peak position of methylene blue, the magnitude of the increase in peak current value measured at-0.28V relative to the current value in the absence of the target molecule is defined as: "Signal Increase%" (I)t-I0)/I0X 100% where It、I0The ACV peak current values at-0.28V were measured in the presence and absence of cocaine in the system, respectively. The results obtained by quantitative detection of cocaine at different concentrations are shown in fig. 7. In the concentration interval of 10 mu mol/L-1 mmol/L, the degree of current increase increases along with the increase of cocaine concentration. The detection limit was 10. mu. mol/L.
Example 5:
the ultramicro gold electrode prepared in the embodiment 1 can be used in the field of electrochemical biosensing, and the specific process for sensing the aptamer comprises the following steps:
(1) assembling the biomolecule marked with electrochemical active substances and the nano flower-shaped ultramicro gold electrode: the ultramicro gold electrode prepared in example 1 was placed in 100nmol/L of dopamine aptamer assembly solution labeled with SH and MB groups, the assembly solution containing 1mol/L NaCl and 20mmol/L MgCl220mmol/L PB, assembled overnight at room temperature protected from light. Wherein, the sequence of the dopamine aptamer is as follows: 5' -SH- (CH)2)6-GTCTCTGTGTGCGCCAGAGACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-MB-3」。
(2) And MCH is sealed: after assembly, the electrode is placed in 2mmol/L MCH solution for reaction for 2h, and the assembly density of the surface probe is regulated and controlled.
(3) Detection of target molecules: and (3) placing the gold electrode prepared in the step (2) in an electrochemical detection solution, performing electrochemical detection in an electrochemical workstation CHI760E, and using a three-electrode system, wherein the working electrode is a nano flower-shaped ultramicro gold electrode, the reference electrode is an Ag/AgCl (3M KCl) electrode, and the counter electrode is a platinum wire electrode. The measurement was carried out by Alternating Current Voltammetry (ACV). The electrochemical workstation "AC voltametry" mode was chosen to detect the electrochemical signal of methylene blue. since-0.28V is the characteristic peak position of methylene blue, the magnitude of the increase in peak current value measured at-0.28V relative to the current value in the absence of the target molecule is defined as: "Signal Increase%" (I)t-I0)/I0X 100% where It、I0The ACV peak current values at-0.28V were measured in the presence and absence of Dopamine (DA) in the system, respectively. The results obtained by quantitative detection of DA at different concentrations are shown in FIG. 8. In the concentration interval of 25 mu mol/L-5 mmol/L, the degree of current increase increases with the increase of DA concentration. In the concentration range of 25-250 μmol/L, the current value increase range and the concentration are in linear relation, and the signal increase% is 0.05092cDAThe linear relation of (mu M) +0.1677 has a correlation coefficient of 0.9575 and the detection limit of DA is 25 mu mol/L.
Claims (3)
1. A preparation method of a nanometer flower-shaped ultramicro gold electrode is characterized by comprising the following steps:
the method comprises the following steps: adhering copper wires and micron-sized carbon fibers with graphite conductive adhesive; the carbon fiber is 7 mu m in diameter and 1.5-2 cm in length, is ultrasonically cleaned for 3min by acetone, ethanol and ultrapure water respectively, is cleaned for 3-5 times by the ultrapure water, and is dried in a constant temperature box at 37 ℃; the length of the copper wire is 8-10 cm;
step two: the copper wire bonded with the carbon fiber axially extends into the glass capillary with the end part pulled up; the diameter of the glass capillary tube is 0.8-1.5 mm, the diameter of the copper wire is smaller than the inner diameter of the glass capillary tube, and the inner diameter of the tip of the drawn glass tube is 50-100 mu m;
step three: the tail part of the capillary tube is sealed by epoxy resin;
step four: the tip of the capillary tube is sealed by flame fusion, and the carbon fiber is exposed out of the tip of the glass capillary tube for flame etching; the flame is the flame of an alcohol lamp, and the tip of the capillary tube is sealed by flame fusing; the diameter of the etched carbon fiber is 100-500nm, and the length of the carbon fiber is 100-300 mu m;
step five: placing the end part of the etched carbon nanofiber into an electrophoretic paint solution for electrochemical deposition; the electrophoretic paint is cathode electrophoretic paint, and the electrophoretic paint solution is obtained by mixing the electrophoretic paint and ultrapure water in a certain proportion and ultrasonically treating; the electrochemical deposition is carried out in a double-electrode system, carbon fibers are used as working electrodes, platinum wires are used as counter electrodes, the deposition voltage is-8V, and the deposition time is 100 s;
step six: heating and baking the electrode on which the electrophoretic paint is deposited; the heating and baking steps are finished in an oven, the temperature is 60-80 ℃, and the baking time is 5-10 min;
step seven: preparing a nano flower-shaped ultramicro gold electrode with a flower-shaped gold nano structure at the end part by electrochemically depositing gold on the treated electrode in an acid chloroauric acid solution; the acid chloroauric acid solution is obtained by mixing hydrochloric acid and chloroauric acid, and the concentration of the hydrochloric acid and the chloroauric acid is 0.5mol/L HCl and 20mmol/L HAuCl4(ii) a The electrochemistryThe deposition is carried out in a three-electrode system, carbon fiber is used as a working electrode, a platinum wire is used as a counter electrode, Ag/AgCl 3M KCl is used as a reference electrode, the deposition voltage is 0V, and the deposition time is 300 s.
2. The nano flower-shaped ultramicro gold electrode prepared by the method of claim 1, wherein the end part of the gold electrode is of a flower-shaped structure, the gold is used as a component, and the diameter of the flower-shaped structure is 50-300 μm.
3. The application of the nano flower-shaped ultramicro gold electrode in the electrochemical biosensing is characterized in that biomolecules marked with electrochemical active substances are assembled on the surface of the prepared gold electrode, the biomolecules are identified with target molecules to cause the change of the distance between the electrochemical active molecules and the surface of the gold, the electrochemical active molecules are detected by an electrochemical workstation, and the electrochemical detection of the target molecules is specifically realized, and the application process is as follows:
(1) assembling the biomolecule marked with electrochemical active substances and the nano flower-shaped ultramicro gold electrode: assembling biomolecules and the surface of a gold electrode through sulfydryl modification, wherein the marked electrochemical active substance is methylene blue or ferrocene, the biomolecules are polypeptide, DNA or aptamer, and the assembling condition is that the biomolecules are assembled overnight at room temperature in a salt solution with certain concentration in a dark place;
(2) and MCH is sealed: placing the assembled electrode in 2mmol/L mercaptohexanol solution for reacting for 2h at room temperature so as to regulate and control the assembly density of the surface probe;
(3) detection of target molecules: placing the gold electrode prepared in the step (2) in an electrochemical detection solution, and measuring electrochemical signals of electrochemical active substances before and after the addition of target molecules by adopting a three-electrode system through Alternating Current Voltammetry (ACV) or Square Wave Voltammetry (SWV);
the target molecule is one of cells, proteins and small molecules.
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| CN110132921B (en) * | 2019-05-16 | 2021-09-14 | 中国科学院重庆绿色智能技术研究院 | Method for monitoring enzyme digestion reaction in real time based on needle tip nanopore monomolecular detection technology |
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| CN112268943B (en) * | 2020-10-14 | 2023-04-07 | 湖南科技大学 | Micro-electrode photoelectrochemical sensor and preparation method and application thereof |
| CN113325059A (en) * | 2021-05-26 | 2021-08-31 | 辽宁师范大学 | Electrochemical detection device for measuring dopamine by virtue of micro-volume selectivity |
| CN113325054B (en) * | 2021-05-31 | 2022-03-01 | 浙江大学 | Fully-integrated portable carbon fiber microelectrode electrochemical sensor and detection system |
| CN113406162B (en) * | 2021-06-03 | 2022-10-21 | 浙江大学 | Preparation method for forming nano-gap electrode pair |
| CN117074488B (en) * | 2023-10-17 | 2024-04-09 | 北京科技大学 | Ultramicro electrode for high-temperature molten salt system test and preparation method and application thereof |
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