Nanoprobes integrating in situ internal reference, dosing and site-specific recording and methods of making

Abstract

The application provides a kind of integrated in-situ internal reference, nano probe of dosing and fixed point recording and preparation method, and the preparation method comprises the following steps: inserting a metal wire into the middle of the length direction of one channel of a double-channel glass tube, heating and melting, and then drawing a double-channel nano tip; sputtering a metal conductive layer on the surface of the double-channel nano tip, depositing a functional layer, connecting the functional layer outer surface and the metal channel lead, and depositing a protective layer; using atmospheric pressure micro-plasma jet to controllably remove the protective layer of the tip, so that the functional layer is slightly exposed; using micro-plasma jet to remove the exposed functional layer tip, so that the metal channel and the dosing channel are exposed; placing the double-channel nano tip in a pre-set solution to form an internal reference electrode in the metal channel. The probe prepared by the application can simultaneously realize in-situ internal reference, drug delivery and reliable fixed point recording of specific signals in a single cell, which is of great significance for accurate diagnosis, real-time monitoring and in-situ treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a nanoprobe integrating in-situ internal reference, drug administration and fixed-point recording, and a preparation method thereof. Background Art

[0002] The development of intracellular implantable neural probes is of great significance to the study of brain-computer interaction and neuroscience. Nanoprobes modified with nanoparticles have minimal implantation damage, high spatiotemporal resolution, and targeted selectivity. They can record specific signals within single neurons in real time, providing a new technical means for in-depth and comprehensive research on brain science and brain diseases. The performance of implantable nanoprobes depends on their ability to transfer charge between the exposed area of ​​the patch clamp probe tip and specific regions within the cell. Voltage, current, and electrochemical methods can be used to quantitatively study the mechanisms of cellular activity in ultramicro or nanoscale spaces, such as vesicle content, membrane potential changes, and the distribution of intracellular substances.

[0003] However, existing nanoprobes typically rely on external references during recording and lack effective tip protection, which affects the sensitivity, targeting, stability, and fidelity of signal detection. Furthermore, nanoprobes without in situ injection capabilities cannot accurately assess the targeted intervention effects of drug delivery on intracellular biomarkers. Currently known nanoprobes are unable to simultaneously achieve intracellular in situ internal reference, in situ drug delivery, and reliable fixed-point recording.

[0004] After searching the prior art, we found:

[0005] The Chinese invention patent application publication number CN111661814A discloses a multifunctional integrated ultramicro / nano electrode and a preparation method thereof. The electrode is prepared based on a drawing method. A tubular mold is filled with liquid low-melting-point metal, and a tube for preparing a functional channel is inserted into the mold. After the low-melting-point metal solidifies, the mold is removed. A solid low-melting-point metal block wrapped with multiple or multiple tubes for preparing different functional channels can be inserted into the middle of a glass tube. The low-melting-point metal is heated to melt and solidify, and then adhered to the inner wall of the glass tube. A probe is prepared by a drawing method, and the tip of the probe is polished. The low-melting-point metal at the front end of the probe is removed. This method can realize the multifunctional and multi-channel integration of multiple electrode recording points, liquid channels, and optical paths. Functional channels of different types, quantities, and geometric parameters can be integrated according to specific applications, breaking away from the limitation of the number of functional channels on the number of holes in the porous glass tube in the traditional drawing method. However, the patent still has the following problems: (1) The probe cannot realize in-situ internal reference and sensing markers such as ions, molecules and organic matter; (2) This method uses a reactive ion etcher to remove the polymer material to expose the internal metal block. Compared with the atmospheric pressure microplasma jet processing method, it is impossible to control the exposure degree of the tip, so it is impossible to obtain a nanoprobe with a fixed-point recording function; (3) This method uses a needle grinder to grind and polish the tip of the drawn probe. Compared with the atmospheric pressure microplasma jet processing, it is impossible to realize the controllable exposure of the internal functional material. It can only grind the tip to form a disk electrode structure, which will result in an excessively small contact area with the substance to be detected, affecting the signal-to-noise ratio and sensitivity of the detection.

[0006] The Chinese invention patent application publication number CN114228218A discloses an implantable flexible, high-density, multifunctional neural probe and a preparation method thereof. The probe uses a plurality of polymers to prepare a first composite polymer preform, which is drawn into filaments by a hot drawing method and a number of filaments are cut; the prepared composite polymer filaments and a plurality of polymers are then used to prepare a second composite polymer preform, which is drawn into filaments by a hot drawing method and a number of filaments are cut; the preform preparation and hot drawing can be repeated multiple times, and the end face of one end of the filament finally obtained is ground and polished, cleaned, and dried. The flexible neural probe prepared by the hot drawing method of the invention can realize the multifunctional and multi-channel integration of probe recording points, liquid channels, and optical pathways. Functional channels of different types, quantities, and geometric parameters can be freely and flexibly integrated according to specific applications. At the same time, by grinding and polishing at different angles, stimulation or signal acquisition can be achieved on the same plane or at different depths. However, the patent still has the following problems: (1) The polymer neural probe has poor stiffness, and the tip size of the polymer neural probe drawn by thermal drawing is difficult to reach the nanometer level, which cannot be used for intracellular implantation detection; (2) The probe can achieve stimulation, drug delivery and signal acquisition of neural tissue in the same plane or at different depths, but because it cannot achieve controllable exposure of the needle tip, it does not have the ability to deliver drugs in situ and record at a fixed point; (3) Since the probe does not have an integrated internal reference and is not effectively protected by a protective layer on the outside, it affects the targeting and fidelity of the recorded signal.

[0007] Therefore, there is an urgent need to propose an implantable nanoprobe that can simultaneously achieve intracellular in situ internal reference, in situ drug delivery and reliable fixed-point recording, which is of great significance for the accurate diagnosis, real-time monitoring and in situ treatment of neurological diseases. Summary of the Invention

[0008] In view of the defects in the prior art, the purpose of the present invention is to provide a nanoprobe and a preparation method thereof that integrates in-situ internal reference, drug delivery and fixed-point recording.

[0009] According to one aspect of the present invention, a method for preparing a nanoprobe integrating in situ internal reference, drug delivery and fixed-point recording is provided, comprising:

[0010] Inserting a metal wire into the middle of one of the channels in the double-channel glass tube in the length direction, heating and melting it, and then drawing it to form a double-channel nanotip, wherein the double-channel nanotip includes a metal channel and a drug delivery channel;

[0011] sputtering metal on the surface of the dual-channel nanotip to form a conductive layer, depositing a functional layer, drawing wires from the outer surface of the functional layer and the metal channel, and depositing a protective layer;

[0012] Using atmospheric pressure micro plasma jet to controllably remove the protective layer of the tip, so that the functional layer is slightly exposed, and fixed-point recording is achieved;

[0013] A microplasma jet is used to remove the exposed tip of the functional layer, exposing the metal channel and drug delivery channel of the dual-channel nanotip, thus achieving drug delivery function.

[0014] The dual-channel nanotip is placed in a preset solution to form an internal reference electrode in the metal channel to achieve an internal reference function.

[0015] Furthermore, the outer diameter of the dual-channel glass tube is 0.2-2 mm, and the inner diameter is 0.1-1.5 mm; the material of the metal wire is any one of silver, mercury and copper, the length is 0.2-50 mm, and the outer diameter is 0.1-1 mm; the diameter of the dual-channel nanotip is 1-1000 nm.

[0016] Furthermore, the material of the conductive layer is any one or more of chromium, gold, silver, platinum, aluminum and iridium; the material of the functional layer is a material sensitive to any one of ions, molecules and organic matter; the material of the protective layer is any one of polyparaxylene, paraffin, aluminum oxide, silicon dioxide, silicon carbide, diamond-like carbon and diamond.

[0017] Furthermore, leads are respectively led from the outer surface of the functional layer and the metal channel, wherein: the leads are two conductive metal wires, one of which is connected to the metal in the dual-channel glass tube through conductive silver paste, and the other is connected to the outer surface of the functional layer through conductive silver paste.

[0018] Furthermore, before sputtering metal to form a conductive layer, the method includes: sputtering a base layer on the surface of the dual-channel nanotip.

[0019] Furthermore, the protective layer of the tip is controllably removed by using an atmospheric pressure microplasma jet, wherein the exciting gas of the atmospheric pressure microplasma jet is a mixed gas of one of helium, argon, and nitrogen and oxygen, wherein the proportion of oxygen is 1-10%.

[0020] Furthermore, the protective layer of the tip is controllably removed by using an atmospheric pressure microplasma jet, wherein the diameter of the atmospheric pressure microplasma jet at its thinnest point is 0.01-1000 μm.

[0021] Furthermore, the exposed functional layer tip is removed by using a microplasma jet, wherein the microplasma jet treatment time is 0-100s.

[0022] Furthermore, the dual-channel nanotip is placed in a preset solution to form an internal reference electrode in the metal channel, wherein the preset solution is any one of saturated potassium chloride solution, hydrochloric acid solution, sulfuric acid solution and potassium sulfate solution, and the internal reference electrode is any one of silver / silver chloride, copper / copper sulfate and mercury / mercurous sulfate.

[0023] According to another aspect of the present invention, a nanoprobe integrating in situ internal reference, drug administration and fixed-point recording is provided, which is prepared using the above-mentioned method for preparing a nanoprobe integrating in situ internal reference, drug administration and fixed-point recording.

[0024] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0025] The present invention manufactures a dual-channel nanotip containing metal in one channel, sputters a conductive layer, deposits a functional layer and a protective layer in sequence, and uses a microplasma jet to treat the probe tip to expose the surface functional layer. This can achieve reliable fixed-point recording of specific signals. At the same time, the metal channel and drug delivery channel of the dual-channel nanotip are exposed and placed in a preset solution to form an internal reference electrode. This allows for simultaneous in-situ internal reference, drug delivery, and reliable fixed-point recording of specific signals within a single cell, helping to accurately evaluate the targeted intervention effects of drug delivery on intracellular markers. This is of great significance for the accurate diagnosis, real-time monitoring, and in situ treatment of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0027] Figure 1 Schematic diagram of a process for preparing a nanoprobe integrating in-situ internal reference, drug delivery, and fixed-point recording in one embodiment of the present invention;

[0028] Figure 2 Schematic diagram of the overall structure of a nanoprobe integrating in-situ internal reference, drug delivery, and fixed-point recording in one embodiment of the present invention;

[0029] Figure 3 Schematic diagram of the structure of a nanoprobe tip integrating in-situ internal reference, drug delivery, and fixed-point recording in one embodiment of the present invention;

[0030] Figure 4 Schematic diagram of forming an in-situ internal reference by chlorination of a dual-channel nanotip in one embodiment of the present invention.

[0031] The reference numerals in the figure correspond to: 1-nanoprobe, 2-conductive metal wire, 3-dual-channel nanotube, 4-drug delivery channel, 5-internal reference electrode, 6-base layer, 7-conductive layer, 8-functional layer, 9-protective layer. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0033] Reference Figure 1 As shown, an embodiment of the present invention provides a method for preparing a nanoprobe that integrates in-situ internal reference, drug delivery, and fixed-point recording, comprising:

[0034] S1. Inserting a metal wire into the middle of one of the channels in a dual-channel glass tube in the longitudinal direction, heating and melting it, and then drawing it to form a dual-channel nanotip, which includes a metal channel and a drug delivery channel;

[0035] S2, on the surface of the dual-channel nanotip, metal is sputtered in sequence to form a conductive layer and a functional layer is deposited, and wires are drawn from the outer surface of the functional layer and the metal channel, such as Figure 3 The outer surface of the middle functional layer and the metal channel are respectively connected to the conductive metal wire, and a protective layer is deposited;

[0036] S3. Use an atmospheric pressure microplasma jet to controllably remove the protective layer at the tip. By controlling the treatment time of the microplasma jet and the degree of etching of the protective layer at the probe tip during the physical bombardment and chemical reaction process, a small amount of the functional layer is exposed. The specific exposure level is determined according to the cell being recorded. The probe contacts a specific location within the cell, achieving fixed-point recording within the cell.

[0037] S4. Using a microplasma jet to remove the tip of the exposed functional layer, so that the metal channel and drug delivery channel of the dual-channel nanotip are also exposed, realizing the drug delivery function;

[0038] S5. Place the dual-channel nanotip in a preset solution to form an internal reference electrode in the metal channel to realize the internal reference function. Thus, an implantable nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording is obtained. The overall structure of the nanoprobe 1 is as follows: Figure 2 As shown. Figure 3 The tip structure of the nanoprobe 1 is composed of a dual-channel nanotube 3, a drug delivery channel 4, an internal reference electrode 5, a base layer 6, a conductive layer 7, a functional layer 8, a protective layer 9, and a conductive metal wire 2 from the inside out, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of organic matter in single cells.

[0039] In some embodiments, in step S1, the outer diameter of the dual-channel glass tube is 0.2-2 mm, the inner diameter is 0.1-1.5 mm, the material of the metal wire is any one of silver, mercury and copper, the length is 0.2-50 mm, the outer diameter is 0.1-1 mm, and the diameter of the dual-channel nanotip is 1-1000 nm, wherein the outer diameter of the metal wire is less than half of the inner diameter of the glass tube to ensure smooth insertion. The length of the metal wire can be arbitrarily selected within this range to ensure that there is molten metal in the middle of one channel in the glass tube after heating; the diameter of the dual-channel nanotip is 1-1000 nm, which is specifically related to the size of the cell to be recorded. For example, for a cell with a diameter of 10 μm, the diameter of the dual-channel nanotip should be less than 300 nanometers to ensure that the probe can successfully break the membrane and enter the cell.

[0040] In some embodiments, in step S2, the material of the conductive layer is any one or more of chromium, gold, silver, platinum, aluminum and iridium, etc., depending on the purpose of the conductive layer. For example, a chromium layer can be selected to enhance the bonding strength between the glass tube and the functional layer, a gold layer can be selected to enhance the conductivity, and an iridium layer can be selected to grow iridium oxide; the material of the functional layer is a material sensitive to any one of ions, molecules and organic matter, for example, iridium oxide sensitive to hydrogen ions, platinum black sensitive to active oxygen, and graphene sensitive to dopamine; the material of the protective layer is any one of polyparaxylene, paraffin, aluminum oxide, silicon dioxide, silicon carbide, diamond-like carbon and diamond, etc., so that the recording of the exposed site of the probe tip is not interfered with by environmental factors.

[0041] In the above embodiment, the leads are two conductive metal wires, one of which is connected to the metal in the double-channel glass tube through conductive silver paste, and the other is connected to the outer surface of the functional layer through conductive silver paste.

[0042] In some embodiments, in step S2, before sputtering metal to form a conductive layer, the step includes: sputtering a primer layer on the surface of the dual-channel nanotip to enhance the bonding effect of the conductive layer.

[0043] In some embodiments, in step S3, an atmospheric pressure microplasma jet is used to controllably remove the protective layer from the tip. The excitation gas for the atmospheric pressure microplasma jet is a mixture of one of helium, argon, nitrogen, and the like, and oxygen, wherein the oxygen accounts for 1-10%. The diameter of the atmospheric pressure microplasma jet at its finest point is 0.01-1000 μm, determined by the inner diameter of the plasma jet generating nozzle. Depending on the diameter of the probe tip and the thickness of the protective layer, a nozzle with a smaller inner diameter can produce a finer jet, resulting in a probe tip with a smaller diameter and a thinner protective layer.

[0044] In some embodiments, in step S4, the microplasma jet processing time is 0-100s, which is determined by the difficulty of etching the protective layer. The more difficult the protective layer is to etch, the longer the processing time. If the processing time range is exceeded, the exposed area will be too large, affecting the fixed-point monitoring capability of the probe.

[0045] Figure 4 A schematic diagram illustrates the formation of an in-situ internal reference by chlorination of a dual-channel nanotip. In some embodiments, the dual-channel nanotip is placed in a predetermined solution, such as a saturated potassium chloride solution, a hydrochloric acid solution, a sulfuric acid solution, or a potassium sulfate solution, corresponding to the material of the metal wire used. This ensures that the metal at the tip of the metal-containing channel within the nanotube reacts with the predetermined solution to form a solution such as silver / silver chloride, copper / copper sulfate, or mercury / mercurous sulfate, thereby providing a good reference function.

[0046] Another embodiment of the present invention provides a nanoprobe that integrates in situ internal reference, drug delivery, and fixed-point recording. This probe is prepared using the aforementioned method for preparing a nanoprobe that integrates in situ internal reference, drug delivery, and fixed-point recording. The tip structure of this nanoprobe comprises, from the inside out, a dual-channel nanotube, a drug delivery channel, an internal reference electrode, a base layer, a conductive layer, a functional layer, a protective layer, and a conductive metal wire, thereby enabling in situ internal reference, drug delivery, and reliable fixed-point recording of organic matter within single cells.

[0047] Below in conjunction with specific embodiment, the scheme of the application will be explained.It will be appreciated by those skilled in the art that the following examples are merely used to illustrate the application and should not be considered as limiting the scope of the application.In the embodiment, if no specific technology or conditions are indicated, the technology or conditions described in the document in this area or the product instructions are used. Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained through commercial means.

[0048] Example 1

[0049] This embodiment provides a method for preparing an implantable nanoprobe that integrates in-situ internal reference, drug delivery, and fixed-point recording functions, which is performed according to the following steps:

[0050] S1, refer to Figure 1 As shown in the figure, a silver wire with a length of 5 mm and an outer diameter of 0.3 mm is inserted into the middle of a channel in a double-channel quartz glass tube with an outer diameter of 1 mm and an inner diameter of 0.5 mm, heated and melted, and then pulled into a double-channel nanotip with a diameter of 100 nm with the assistance of a laser.

[0051] S2, reference Figure 1As shown, a chromium base layer and a gold conductive layer are sputtered onto the tip of the dual-channel nanotube. A functional layer sensitive to organic matter is then deposited. Two conductive metal wires, here enameled wires with polished ends, are then introduced: one connected to the silver in the dual-channel glass tube, the other to the outer surface of the functional layer. Subsequently, a protective layer of parylene 9 is chemically vapor deposited.

[0052] S3, refer to Figure 1 As shown, an atmospheric-pressure microplasma jet of a helium-oxygen mixture controllably removes the protective layer from the probe tip, exposing a tiny amount of the functional layer and enabling pinpoint recording of specific signals. The helium flow rate is 60 sccm, the oxygen flow rate is 3 sccm, and the diameter of the thinnest jet is 1 μm.

[0053] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, exposing the silver channel and drug delivery channel at the tip of the dual-channel nanotube, thereby realizing the drug delivery function. The microplasma jet treatment time is 2s.

[0054] S5, refer to Figure 1 and Figure 4 As shown, the dual-channel nanotip is placed in a saturated potassium chloride solution to chlorinate the silver-containing channel, forming a silver / silver chloride internal reference, thus realizing the internal reference function. Figure 2 , to obtain implantable nanoprobes, refer to Figure 3 The tip structure of the nanoprobe is composed of a dual-channel nanotube, a drug delivery channel, a silver / silver chloride internal reference electrode, a chromium base layer, a gold conductive layer, a functional layer sensitive to organic matter, a polyparaxylene protective layer, and an enameled conductive metal wire from the inside out, thus realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of organic matter in single cells.

[0055] Example 2

[0056] This embodiment provides a method for preparing an implantable nanoprobe that integrates in-situ internal reference, drug delivery, and fixed-point recording functions, which is performed according to the following steps:

[0057] S1, refer to Figure 1 As shown in the figure, mercury with a length of 2 mm and an outer diameter of 0.5 mm was injected into the middle of a channel in a double-channel borosilicate glass tube with an outer diameter of 1.5 mm and an inner diameter of 1 mm, and then pulled into a double-channel nanotip with a diameter of 200 nm with the assistance of a laser.

[0058] S2, reference Figure 1As shown, a chromium base layer and a platinum conductive layer are sputtered onto the tip of the dual-channel nanotube. An ion-sensitive functional layer is then deposited. Two copper conductive wires are then connected: one to the mercury in the dual-channel glass tube and the other to the outer surface of the functional layer. Subsequently, a diamond-like carbon protective layer is deposited via plasma vapor deposition.

[0059] S3, refer to Figure 1 As shown, an atmospheric-pressure microplasma jet of a mixture of argon and oxygen is used to controllably remove the protective layer from the probe tip, exposing a tiny amount of the functional layer and enabling the pinpoint recording of specific signals. The argon flow rate is 40 sccm, the oxygen flow rate is 1 sccm, and the diameter of the thinnest jet is 0.5 μm.

[0060] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, exposing the mercury channel and drug delivery channel at the tip of the dual-channel nanotube, thereby realizing the drug delivery function. The microplasma jet treatment time is 1 s.

[0061] S5, refer to Figure 1 and Figure 4 As shown, the dual-channel nanotip is placed in a saturated potassium sulfate solution, so that the channel containing mercury forms a mercury / mercury sulfate internal reference electrode, realizing the internal reference function. Figure 2 , to obtain implantable nanoprobes, refer to Figure 3 The tip structure of the nanoprobe is composed of a dual-channel nanotube, a drug delivery channel, a mercury / mercuric sulfate internal reference electrode, a chromium base layer, a platinum conductive layer, an ion-sensitive functional layer, a diamond-like carbon protective layer, and a copper conductive metal wire from the inside out, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of ions in single cells.

[0062] Example 3

[0063] This embodiment provides a method for preparing an implantable nanoprobe that integrates in-situ internal reference, drug delivery, and fixed-point recording functions, which is performed according to the following steps:

[0064] S1, refer to Figure 1 As shown, a silver wire with a length of 3 mm and an outer diameter of 0.8 mm was inserted into the middle of a channel in a double-channel quartz glass tube with an outer diameter of 2 mm and an inner diameter of 1.5 mm. After heating and melting, it was directly pulled into a double-channel nanotip with a diameter of 500 nm.

[0065] S2, reference Figure 1 As shown, a chromium base layer and an iridium conductive layer are sputtered onto the tip of the dual-channel nanotube. A molecule-sensitive functional layer is then deposited. Two silver conductive wires are then connected: one to the silver inside the dual-channel glass tube and the other to the outer surface of the functional layer. Subsequently, a silicon dioxide protective layer is deposited using chemical vapor deposition.

[0066] S3, refer to Figure 1 As shown, an atmospheric-pressure microplasma jet of a helium-oxygen mixture controllably removes the protective layer from the probe tip, exposing a tiny amount of the functional layer and enabling pinpoint recording of specific signals. The helium flow rate is 20 sccm, the oxygen flow rate is 0.8 sccm, and the diameter of the thinnest jet is 1.2 μm.

[0067] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, exposing the silver channel and the drug delivery channel at the tip of the dual-channel nanotube 3, thereby realizing the drug delivery function. The microplasma jet treatment time is 2.5 s.

[0068] S5, refer to Figure 1 and Figure 4 As shown, the dual-channel nanotip is placed in a hydrochloric acid solution to chlorinate the silver-containing channel, forming a silver / silver chloride internal reference, thus realizing the internal reference function. Figure 2 , the prepared implantable nanoprobe was obtained, referring to Figure 3 The tip structure of the nanoprobe is composed of a dual-channel nanotube, a drug delivery channel, a silver / silver chloride internal reference electrode, a chromium base layer, an iridium conductive layer, a molecule-sensitive functional layer, a silicon dioxide protective layer, and a silver conductive metal wire from the inside out, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of molecules in single cells.

[0069] The above embodiment of the present invention manufactures a dual-channel nanotip containing metal in one channel, sputters metal in sequence to form a conductive layer, deposits a functional layer and a protective layer, and uses a microplasma jet to treat the probe tip to expose the surface functional layer, thereby achieving reliable point-to-point recording of specific signals. At the same time, the metal channel and drug delivery channel of the dual-channel nanotip are exposed and placed in a preset solution to form an internal reference electrode, thereby having the function of simultaneously realizing in situ internal reference, drug delivery and reliable point-to-point recording of specific signals in a single cell, which helps to accurately evaluate the targeted intervention effect of drug delivery on intracellular markers, which is of great significance for the accurate diagnosis, real-time monitoring and in situ treatment of various diseases.

[0070] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various modifications or variations within the scope of the claims without affecting the essence of the present invention. The above preferred features may be used in any combination as long as they do not conflict with each other.

Claims

1. A method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording, characterized in that: include: Inserting a metal wire into the middle of one of the channels in the double-channel glass tube in the length direction, heating and melting it, and then drawing it to form a double-channel nanotip, wherein the double-channel nanotip includes a metal channel and a drug delivery channel; sputtering metal on the surface of the dual-channel nanotip to form a conductive layer, depositing a functional layer, drawing wires from the outer surface of the functional layer and the metal channel, and depositing a protective layer; Using atmospheric pressure micro plasma jet to controllably remove the protective layer of the tip, so that the functional layer is slightly exposed, and fixed-point recording is achieved; A microplasma jet is used to remove the exposed tip of the functional layer, exposing the metal channel and drug delivery channel of the dual-channel nanotip, thus achieving drug delivery function. The dual-channel nanotip is placed in a preset solution to form an internal reference electrode in the metal channel to achieve an internal reference function.

2. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The outer diameter of the dual-channel glass tube is 0.2-2 mm, and the inner diameter is 0.1-1.5 mm; the material of the metal wire is any one of silver, mercury and copper, with a length of 0.2-50 mm and an outer diameter of 0.1-1 mm; the diameter of the dual-channel nanotip is 1-1000 nm.

3. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The material of the conductive layer is any one or more of chromium, gold, silver, platinum, aluminum and iridium; the material of the functional layer is a material sensitive to any one of ions, molecules and organic matter; the material of the protective layer is any one of polyparaxylene, paraffin, aluminum oxide, silicon dioxide, silicon carbide, diamond-like carbon and diamond.

4. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: Leads are respectively led from the outer surface of the functional layer and the metal channel, wherein: the leads are two conductive metal wires, one of which is connected to the metal in the dual-channel glass tube through conductive silver paste, and the other is connected to the outer surface of the functional layer through conductive silver paste.

5. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: Before sputtering metal to form a conductive layer, the method includes: sputtering a base layer on the surface of the dual-channel nanotip.

6. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The method uses an atmospheric pressure microplasma jet to controllably remove the protective layer of the tip, wherein the exciting gas of the atmospheric pressure microplasma jet is a mixed gas of one of helium, argon, and nitrogen and oxygen, wherein the proportion of oxygen is 1-10%.

7. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The atmospheric pressure micro plasma jet is used to controllably remove the protective layer of the tip, wherein the diameter of the atmospheric pressure micro plasma jet at its thinnest point is 0.01-1000 μm.

8. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The exposed functional layer tip is removed by using a microplasma jet, wherein the microplasma jet treatment time is 0-100s.

9. The method for preparing a nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording according to claim 1, characterized in that: The dual-channel nanotip is placed in a preset solution to form an internal reference electrode in the metal channel, wherein the preset solution is any one of a saturated potassium chloride solution, a hydrochloric acid solution, a sulfuric acid solution, and a potassium sulfate solution, and the internal reference electrode is any one of silver / silver chloride, copper / copper sulfate, and mercury / mercurous sulfate.

10. A nanoprobe integrating in-situ internal reference, drug delivery and fixed-point recording, characterized in that: The nanoprobe is prepared by the preparation method of any one of claims 1 to 9 that integrates in-situ internal reference, drug administration and fixed-point recording.

Images