Preparation of gold microsphere glass tube hybrid electrode and application of gold microsphere glass tube hybrid electrode in electrochemical luminescence imaging

Abstract

The invention discloses a preparation method of a gold microsphere glass tube hybrid electrode and an electrochemical luminescence imaging application of the gold microsphere glass tube hybrid electrode. According to the method, microspheres of any size and material are used as raw materials, and a high-repeatability microelectrode is prepared. By means of a soft substrate polymer and a gold microsphere array, microspheres and a microtube electrode are combined through an accurate micromanipulation technology, and a stable hemispherical microelectrode structure is constructed. The method is suitable for microspheres of various sizes, and microspheres of corresponding materials can be selected according to different application requirements. In addition, the preparation method has high repeatability and controllability, and can ensure that the performance of the microelectrode prepared each time is highly consistent. Therefore, the microelectrode prepared by the method has a wide application prospect in the fields of biosensing, micro / nano processing and the like.

Description

Technical Field

[0001] The present invention belongs to the field of microelectrode preparation, and particularly relates to a preparation method of a gold microsphere glass tube hybrid electrode and its application in electrochemiluminescence imaging. Background Art

[0002] Self-assembly technology and chemical plating technology provide new ways for the preparation of microelectrodes with specific functions. Traditional preparation methods mainly include electrodeposition method and electrochemical etching method. The electrochemical deposition method forms a hemispherical structure on the surface of the microelectrode by precisely controlling the potential and electrolyte composition, but the difficulty lies in the precise regulation of the deposition rate and solution composition. Lithography and etching technology define patterns by lithography and form structures by etching, but it is limited by lithography resolution and etching uniformity, and the cost of high-precision equipment is relatively high. In addition to these traditional methods, 3D printing technology can directly prepare hemispherical microelectrodes, but its printing accuracy and material selection are limited, and it is difficult to meet the requirements of micron-level accuracy. These technologies provide new ways for the rapid preparation of microelectrodes. The development of these innovative methods not only improves the preparation efficiency of microelectrodes, but also provides more possibilities for the application of microelectrodes in the fields of analytical chemistry, biology and medicine.

[0003] A microelectrode is a precision electrode with a size not exceeding 25 μm, and has characteristics such as high current density, fast response, low IR drop and high signal-to-noise ratio. These advantages make it perform excellently in electrochemical tests and are widely used in the fields of analytical chemistry, biology and medicine. Especially in the field of life sciences, such as single-cell detection and in vivo analysis, microelectrodes play an indispensable role and provide precise measurement tools for research.

[0004] Therefore, it is of great significance to develop a preparation method of a single hemispherical microelectrode with simple operation, low cost, not limited by material properties and high signal-to-noise ratio. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a preparation method of a gold microsphere glass tube hybrid electrode. This method can precisely assemble a single microsphere into a glass micropipette, which is not only simple to operate, but also has a low cost, and has significant advantages compared with the previous preparation methods.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A preparation method of a gold microsphere glass tube hybrid electrode, the specific steps are as follows:

[0008] Step (1) Immerse the borosilicate glass capillary tube into pure acetone, pure ethanol and ultrapure water in sequence, and perform ultrasonic treatment for 30 min respectively to remove the surface residues; then, use N 2Dry the capillary surface and place it in an oven at 60 °C for 120 min of drying treatment; Pass through CO 2 Use a CO 2 laser puller to draw the dried capillary into a micropipette with a tip inner diameter of 3.1 - 3.2 μm;

[0009] In step (2), use a high - vacuum magnetron ion sputtering instrument to perform a gold layer sputtering treatment on the micropipette prepared in step (1); During the sputtering process, the gold layer can be uniformly deposited on the inner surface of the pipette tip and the entire outer surface, thereby obtaining a microtube electrode coated with a gold layer;

[0010] In step (3), pour the mixed soft - substrate polymer solution onto a template with a 3 - μm pore pattern and cure it to prepare a hydrophobic soft substrate; After curing, cut the prepared porous soft substrate into 2 cm 2 × 2 cm 2 and fix it on a glass slide;

[0011] In step (4), wash the 3.3 - μm gold microspheres 3 times with pure ethanol to thoroughly remove the impurities on their surfaces; After washing, redisperse the gold microspheres in a pure ethanol solution, and then uniformly drop them onto the surface of the soft substrate in step (3); After natural air - drying, a uniformly distributed gold microsphere array is formed;

[0012] In step (5), install the gold - layer - coated microtube electrode prepared in step (2) on a micromanipulator; By adjusting the R - axis of the micromanipulator, accurately control the angle between the microtube electrode and the soft substrate; With the assistance of a microscope, accurately locate the gold microsphere array in step (4); Subsequently, with the help of the X, Y, and Z three - axis fine - tuning function of the micromanipulator, slowly move the microtube tip closer to one end of the target microsphere; When the microtube tip approaches the target microsphere, lower the Z - axis so that the open end of the microtube gently presses on the soft substrate, ensuring that the center of the microtube orifice is on the same horizontal plane as the microsphere; Then, adjust the X - axis to insert part of the microsphere into the microtube electrode; Finally, raise the Z - axis to pick up a single microsphere from the soft substrate and transfer it to the microtube tip to obtain a microsphere electrode supported by a microtube;

[0013] In step (6), dissolve the sealing lubricating wax in chloroform solvent and perform ultrasonic treatment for 30 min to ensure that the lubricating wax is completely dissolved; Subsequently, immerse the microsphere electrode prepared in step (5) in the lubricating wax solution, take it out and keep it in a vertical state to dry naturally, allowing the chloroform solvent to fully volatilize; Repeat the above dipping and drying steps twice to ensure that the surface of the microelectrode is evenly covered with lubricating wax, thereby completing the sealing treatment of the microelectrode; Thus, the hemispherical microelectrode is prepared.

[0014] As an improvement of the present invention, in the step (1), P - 2000CO 2The laser drawing instrument is used to draw capillary tubes, and the specific parameter settings are HEAT = 300, FIL = 5, VEL = 45, DEL = 160, PUL = 0.

[0015] As an improvement of the present invention, in the step (2), an Au target is used, and the tip of the micropipette is placed perpendicular to the sputtering source, and sputtering is carried out for 30 min with a current of 100 mA.

[0016] As an improvement of the present invention, in the step (3), the curing temperature is 70 °C and the curing time is 60 min.

[0017] As an improvement of the present invention, in the step (5), the angle between the microtube electrode and the soft substrate is 5° - 10°.

[0018] As an improvement of the present invention, in the step (6), 1 cm 3 The sealing lubricating wax is dissolved in 2 mL of chloroform to prepare a nearly saturated solution.

[0019] The present invention also provides an application of the microelectrode prepared by the above preparation method in electrochemiluminescence imaging.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention successfully prepares a preparation method of a gold microsphere glass tube hybrid electrode, namely a hemispherical microelectrode, by using a soft substrate, and combines a single microsphere with a gold-plated micropipette to realize the construction of a micron-scale electrode. The use of the soft substrate enables the micromanipulator to accurately pick up any microsphere on the substrate, thus avoiding the randomness problem in the traditional method. This precise manipulation ability not only improves the controllability and repeatability of microelectrode preparation, but also provides higher precision and flexibility for the construction of micron-scale electrodes.

[0022] 2. The present invention combines a single microsphere with a micropipette to propose a new method for preparing a hemispherical microelectrode. This method has the advantages of high-efficiency preparation, simple operation, low cost, etc., can significantly improve the yield, reduce the operation difficulty and material consumption, and effectively solve the problems of low yield and complex process in the prior art. The present invention provides an efficient, simple and economical preparation scheme for hemispherical microelectrodes, which has broad application prospects. Description of the Drawings

[0023] Figure 1 It is a schematic diagram of the preparation of the gold microsphere glass tube hybrid electrode of the present invention;

[0024] Figure 2 It is a scanning electron microscope image of the gold microsphere glass tube hybrid electrode of the present invention;

[0025] Figure 3 Cyclic voltammetry curve of the gold microsphere glass tube hybrid electrode of the present invention in KHP with 1 mM FeMeOH and 0.1 M KCl;

[0026] Figure 4 Bright-field image, electrochemiluminescence imaging image of the gold microsphere glass tube hybrid electrode, and electrochemiluminescence intensity change curve along the axial direction of the microelectrode. Detailed implementation manners

[0027] The present invention will be further clarified below in conjunction with the accompanying drawings and detailed implementation manners. It should be understood that the following detailed implementation manners are only used to illustrate the present invention and not to limit the scope of the present invention. It should be noted that the terms "front", "rear", "left", "right", "upper" and "lower" used in the following description refer to the directions in the accompanying drawings, and the terms "inner" and "outer" respectively refer to the directions towards or away from the geometric center of a specific component.

[0028] Example 1: Preparation method of the gold microsphere glass tube hybrid electrode.

[0029] Figure 1 The preparation method of the gold microsphere glass tube hybrid electrode of the present invention includes the following steps:

[0030] (1) Immerse the borosilicate glass capillary tube successively in pure acetone, pure ethanol and ultrapure water, and perform ultrasonic treatment for 30 min respectively to remove the surface residues; subsequently, use N 2 to blow dry the surface of the capillary tube, and place it in an oven at 60 °C for drying treatment for 120 min; use a CO 2 laser puller to pull the dried capillary tube into a micropipette with a tip inner diameter of 3.1 - 3.2 μm;

[0031] (2) Use a high-vacuum magnetron ion sputtering instrument to perform gold layer sputtering treatment on the micropipette prepared in step (1); during the sputtering process, the gold layer can be evenly deposited on the inner surface of the tip of the pipette and the entire outer surface, thereby obtaining a microtube electrode coated with a gold layer;

[0032] (3) Pour the mixed soft substrate polymer solution onto a template with a 3-μm pore pattern and cure it. Thus, a hydrophobic soft substrate is prepared; after curing, cut the obtained porous soft substrate into 2 cm 2 × 2 cm 2 and fix it on a glass slide;

[0033] (4) Wash the 3.3-μm gold microspheres three times with absolute ethanol to thoroughly remove the impurities on their surfaces; after the washing is completed, redisperse the gold microspheres in an absolute ethanol solution, and then evenly drop them onto the surface of the soft substrate in step (3); after natural air drying, a uniformly distributed gold microsphere array is formed;

[0034] (5) Mount the microtube electrode coated with a gold layer prepared in step (2) on a micromanipulator; precisely control the angle between the microtube electrode and the soft substrate by adjusting the R axis of the micromanipulator; with the assistance of a microscope, accurately locate the gold microsphere array in step (4); subsequently, by means of the fine adjustment function of the X, Y, and Z axes of the micromanipulator, slowly bring the tip of the microtube close to one end of the target microsphere; when the tip of the microtube approaches the target microsphere, lower the Z axis so that the open end of the microtube gently presses on the soft substrate to ensure that the center of the microtube orifice is on the same horizontal plane as the microsphere; then, adjust the X axis to insert part of the microsphere into the interior of the microtube electrode; finally, raise the Z axis to pick up a single microsphere from the soft substrate and transfer it to the tip of the microtube to obtain a microsphere electrode supported by the microtube;

[0035] (6) Dissolve the sealing lubricating wax in chloroform solvent and perform ultrasonic treatment for 30 min to ensure that the lubricating wax is completely dissolved; subsequently, immerse the microsphere electrode prepared in step (5) in the lubricating wax solution, take it out and keep it in a vertical state to dry naturally to allow the chloroform solvent to volatilize fully; repeat the above dipping and drying steps twice to ensure that the surface of the microelectrode is evenly covered with lubricating wax, thereby completing the sealing treatment of the microelectrode; thus, the hemispherical microelectrode is prepared.

[0036] Example 2: Application of the gold microsphere glass tube hybrid electrode in electrochemiluminescence imaging:

[0037] Ru(bpy) 3 2+ is an electrochemiluminescent substance. To verify whether the prepared gold microsphere glass tube hybrid electrode is successful, an electrochemiluminescence test was carried out on it using a three-electrode system. The hemispherical microelectrode was used as the working electrode, an Ag / AgCl wire was used as the reference electrode, and a platinum wire was used as the counter electrode. The applied potential was +1.0 V (vs Ag / AgCl, 3 M KCl), and at the same time, relevant optical images were collected using an upright optical microscope (brand Nikon, model ECLIPSE Ni-u) equipped with an electron multiplying charge-coupled device (EMCCD, brand Andor, model iXon Ultra 897).

[0038] Figure 2 is a scanning electron microscope image of the gold microsphere glass tube hybrid electrode. The gold microspheres at the electrode tip are exposed, and the tip of the glass tube is coated with sealing lubricating wax.

[0039] Figure 3Cyclic voltammetry curves of the gold microsphere-glass tube hybrid electrode in 1 mM FeMeOH and 0.1 M KCl in KHP. The hemispherical microelectrode has electrical connectivity, electrochemical activity, and stability.

[0040] Figure 4 Bright-field image and electrochemiluminescence imaging image of the gold microsphere-glass tube hybrid electrode. The electrochemiluminescence intensity of the microsphere part of the hemispherical microelectrode is 2200 a.u., respectively, while the light intensity of the main body part of the microtube electrode is the same as the background light intensity.

[0041] The technical means disclosed in the solution of the present invention are not limited to the technical means disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A method for preparing a gold microsphere glass tube hybrid electrode, characterized in that: The specific steps are as follows: Step (1) immersing the borosilicate glass capillary in pure acetone and pure ethanol ultrapure water in turn, and performing ultrasonic treatment for 30 minutes respectively to remove the residue on the surface; Subsequently, the capillary surface was dried with N2 and placed in an oven at 60°C for 120 minutes. The dried capillary was drawn into a micropipette with a tip inner diameter of 3.1-3.2 μm using a CO2 laser drawing instrument. Step (2) using a high vacuum magnetron ion sputtering apparatus to perform a gold layer sputtering treatment on the micropipette prepared in step (1); During the sputtering process, the gold layer can be evenly deposited on the inner surface of the pipette tip and the entire outer surface, thereby obtaining a gold-coated microtube electrode; Step (3) casting the mixed soft substrate polymer solution on a template having a 3 μm pore pattern and curing it to prepare a hydrophobic soft substrate; After curing, the prepared porous soft substrate was cut into 2 cm 2 ×2cm 2 and fix it on a glass slide; Step (4) washing the 3.3 μm gold microspheres three times with pure ethanol to completely remove impurities on the surface thereof; after washing, the gold microspheres are redispersed in a pure ethanol solution, and then evenly added to the surface of the soft substrate in step (3); after natural air drying, an evenly distributed gold microsphere array is formed; Step (5) installing the gold-coated microtube electrode prepared in step (2) on a micromanipulator; by adjusting the R axis of the micromanipulator, accurately controlling the angle between the microtube electrode and the soft substrate; with the assistance of a microscope, accurately positioning the gold microsphere array in step (4); then, using the X, Y, and Z three-axis fine-tuning function of the micromanipulator, slowly bringing the tip of the microtube close to one end of the target microsphere; when the tip of the microtube approaches the target microsphere, lowering the Z axis so that the open end of the microtube is gently pressed against the soft substrate, ensuring that the center of the microtube orifice is in the same horizontal plane as the microsphere; then, adjusting the X axis to partially insert the microsphere into the microtube electrode; finally, raising the Z axis to pick up a single microsphere from the soft substrate and transfer it to the tip of the microtube to obtain a microsphere electrode supported by the microtube; Step (6) dissolving the sealing lubricating wax in chloroform solvent and subjecting to ultrasonic treatment for 30 minutes to ensure that the lubricating wax is completely dissolved; then, immersing the microsphere electrode prepared in step (5) in the lubricating wax solution, taking it out and keeping it in a vertical state to dry naturally, so that the chloroform solvent can fully evaporate; repeating the above-mentioned dipping and drying steps twice to ensure that the surface of the microelectrode is evenly covered with the lubricating wax, thereby completing the sealing treatment of the microelectrode; at this point, the preparation of the hemispherical microelectrode is completed.

2. The method for preparing a gold microsphere-glass tube hybrid electrode according to claim 1, characterized in that: In the step (1), a P-2000 CO2 laser drawing instrument is used to draw the capillary, and the specific parameters used are set as HEAT=300, FIL=5, VEL=45, DEL=160, and PUL=0.

3. The method for preparing a gold microsphere-glass tube hybrid electrode according to claim 1, characterized in that: In the step (2), an Au target material is used, and the tip of a micropipette is placed perpendicular to a sputtering source, and sputtering is performed at a current of 100 mA for 30 minutes.

4. The method for preparing a gold microsphere glass tube hybrid electrode according to claim 1, characterized in that: The curing temperature in step (3) is 70° C. and the curing time is 60 min.

5. The method for preparing a gold microsphere-glass tube hybrid electrode according to claim 1, characterized in that: In the step (5), the angle between the microtube electrode and the soft substrate is 5°-10°.

6. The method for preparing a gold microsphere-glass tube hybrid electrode according to claim 1, characterized in that: In step (6), 1 cm 3 The sealing lubricating wax is dissolved in 2 mL of chloroform to prepare a nearly saturated solution.

7. Application of a microelectrode prepared by the preparation method according to any one of claims 1 to 6 in electrochemiluminescence imaging.

Images