Nanoprobe integrating in-situ internal reference, administration and fixed-point recording and preparation method thereof

Abstract

The invention provides a nano probe integrating in-situ internal reference, drug administration and fixed-point recording and a preparation method thereof, and the preparation method comprises the following steps: inserting a metal wire into the middle of a channel in a dual-channel glass tube in the length direction, heating and melting, and drawing to form a dual-channel nano tip; sputtering a metal conductive layer on the surface of the dual-channel nano tip, depositing a functional layer, leading wires from the outer surface of the functional layer and a metal channel, and depositing a protective layer; the protective layer at the tip is controllably removed by using atmospheric pressure micro plasma jet, so that the functional layer is slightly exposed; removing the exposed tip of the functional layer by using micro plasma jet to expose the metal channel and the administration channel; and placing the dual-channel nano tip in a preset solution to form an internal reference electrode in the metal channel. The probe prepared by the invention can simultaneously realize in-situ internal reference, drug delivery and reliable fixed-point recording of specific signals in single cells, and has important significance on accurate diagnosis, real-time monitoring and in-situ treatment of diseases.

Description

Technical Field

[0001] The 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, and can record specific signals in 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 areas within the cell. The mechanism of cell activity in ultramicro or nanospaces, such as vesicle content, membrane potential changes, and the distribution of intracellular substances, can be quantitatively studied by voltage, current, and electrochemical methods.

[0003] However, existing nanoprobes usually rely on external references during recording and lack effective tip protection, which affects the sensitivity, targeting, stability and fidelity of signal detection. In addition, nanoprobes without in situ injection capabilities cannot accurately evaluate the targeted intervention effects of drug delivery on intracellular biomarkers. Currently known nanoprobes cannot 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 with 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, and 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 is solidified, the mold is removed, and 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 and the low-melting-point metal at the front end of the probe is removed. The multifunctional and multi-channel integration of multiple electrode recording points, liquid channels and optical paths can be realized. Functional channels of different types, quantities and geometric parameters can be integrated according to specific applications, and the number of functional channels is freed from the limitation of the number of holes in porous glass tubes on the number of functional channels in the traditional drawing method. However, the patent still has the following problems: (1) The probe cannot realize in-situ internal reference, and cannot sense 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 pulled probe. Compared with the atmospheric pressure microplasma jet processing, it is impossible to realize the controllable exposure of the internal functional material. The tip can only be ground flat 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 with 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, and uses a hot drawing method to draw the first composite polymer preform into filaments and cut a number of filaments; then the prepared composite polymer filaments and a plurality of polymers are used to prepare a second composite polymer preform, and the second composite polymer preform is drawn into filaments and cut a number of filaments by a hot drawing method. The preform preparation and hot drawing can be repeated multiple times, and the end face of one end of the filament finally obtained is polished, cleaned, and dried. The invention is based on the flexible neural probe prepared by the hot drawing method, which 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, stimulation or signal acquisition on the same plane or at different depths can be achieved by polishing at different angles. However, the patent still has the following problems: (1) The polymer neural probe has poor stiffness. The tip size of the polymer neural probe drawn by thermal drawing is difficult to reach the nanometer level and cannot be used for intracellular implantation detection; (2) The probe can achieve stimulation, drug administration 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 of in situ drug administration and fixed-point recording; (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 object of the present invention is to provide a nanoprobe and a preparation method thereof that integrates in-situ internal reference, drug administration 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 administration 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 pulling it to form a double-channel nano tip, wherein the double-channel nano tip 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, respectively 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 to achieve fixed-point recording;

[0013] Use microplasma jet to remove the exposed functional layer tip, so that the metal channel and drug delivery channel of the dual-channel nanotip are exposed to achieve 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 nano tip 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 tip of the functional layer is removed by using a micro plasma jet, wherein the micro plasma jet treatment time is 0-100 s.

[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 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.

[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, uses a microplasma jet to process the probe tip, and exposes the surface functional layer, thereby enabling reliable fixed-point recording of specific signals. At the same time, the metal channel and the 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 functions of simultaneously realizing in-situ internal reference, drug delivery and reliable fixed-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. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0028] Figure 2 This is a schematic diagram of the overall structure of a nanoprobe that integrates in-situ internal reference, drug administration and fixed-point recording in one embodiment of the present invention;

[0029] Figure 3 A schematic diagram of the structure of a nanoprobe tip that integrates 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 is described in detail below in conjunction with 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 of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0033] Reference Figure 1 As shown, a method for preparing a nanoprobe integrating in-situ internal reference, drug administration and fixed-point recording provided by one embodiment of the present invention comprises:

[0034] S1. 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 pulling it to form a double-channel nano tip, wherein the double-channel nano tip includes a metal channel and a drug delivery channel;

[0035] S2, sputtering metal on the surface of the dual-channel nano-tip to form a conductive layer and depositing a functional layer, and wiring 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 with conductive metal wires, and a protective layer is deposited;

[0036] S3. Use atmospheric pressure microplasma jet to controllably remove the protective layer of the tip. By controlling the processing time of the microplasma jet, the degree of etching of the protective layer of the probe tip by physical bombardment and chemical reaction is controlled, so that a small amount of the functional layer is exposed. The specific exposure degree is determined according to the recorded cell. The probe contacts the specific position in the cell to achieve fixed-point recording in the cell.

[0037] S4, using a micro plasma 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, placing 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 administration and fixed-point recording is obtained. The overall structure of the nanoprobe 1 is as follows Figure 2 See Figure 3 The tip structure of the nanoprobe 1 is, from the inside out, 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, thereby realizing the functions of in-situ internal reference, drug delivery, and reliable fixed-point recording of organic matter in a single cell.

[0039] In some embodiments, in step S1, the outer diameter of the dual-channel glass tube is 0.2-2mm, the inner diameter is 0.1-1.5mm, the material of the metal wire is any one of silver, mercury and copper, the length is 0.2-50mm, the outer diameter is 0.1-1mm, and the diameter of the dual-channel nanotip is 1-1000nm, wherein the outer diameter of the metal wire is less than half of the inner diameter of the glass tube to ensure smooth insertion, and 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 1-1000nm, which is specifically related to the size of the cell to be recorded, for example, for a cell with a diameter of 10μm, 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 force 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, so that the recording of the exposure site of the probe tip is not interfered by environmental factors.

[0041] In the above embodiment, the lead wires 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, the protective layer of the tip is controllably removed using an atmospheric pressure microplasma jet, wherein: the excitation gas of the atmospheric pressure microplasma jet is a mixed gas of one of helium, argon, nitrogen, etc. and oxygen, wherein the proportion of oxygen is 1-10%. The diameter of the atmospheric pressure microplasma jet at the thinnest part is 0.01-1000 μm, which is determined by the inner diameter of the plasma jet generating nozzle. According to the diameter of the probe tip and the thickness of the protective layer, the nozzle with a smaller inner diameter can excite a finer jet, and obtain a probe tip with a smaller processing 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 is. 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 of the dual-channel nanotip chlorination to form an in-situ internal reference is shown. In some embodiments, the dual-channel nanotip is placed in a preset solution, which is any one of a saturated potassium chloride solution, a hydrochloric acid solution, a sulfuric acid solution, a potassium sulfate solution, etc., corresponding to the material of the metal wire used, to ensure that the metal at the tip of the metal-containing channel in the nanotube reacts with the preset solution to form any one of silver / silver chloride, copper / copper sulfate, mercury / mercurous sulfate, etc., so that it has a good reference function.

[0046] Another embodiment of the present invention provides a nanoprobe integrating in-situ internal reference, drug administration and fixed-point recording, which is prepared by the above-mentioned preparation method of the nanoprobe integrating in-situ internal reference, drug administration and fixed-point recording. The tip structure of the nanoprobe is a dual-channel nanotube, a drug administration channel, an internal reference electrode, a base layer, a conductive layer, a functional layer, a protective layer, and a conductive metal wire from the inside to the outside, thereby realizing the functions of in-situ internal reference, drug delivery and reliable fixed-point recording of organic matter in a single cell.

[0047] The scheme of the present application will be explained below in conjunction with specific embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the documents in this area or the product specifications are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained through commercial channels.

[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, 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 on the tip surface of the dual-channel nanotube, a functional layer sensitive to organic matter is deposited, and two conductive metal wires are introduced, which are enameled wires with polished ends, one connected to the silver in the dual-channel glass tube and the other connected to the outer surface of the functional layer. Subsequently, a polyparaxylene protective layer 9 is chemically vapor deposited.

[0052] S3, refer to Figure 1 As shown in the figure, the atmospheric pressure micro plasma jet mixed with helium and oxygen is used to controllably remove the protective layer at the tip of the probe, exposing a small amount of the functional layer and realizing the fixed-point recording function of specific signals. The flow rate of helium is 60sccm, the flow rate of oxygen is 3sccm, and the diameter of the thinnest part of the jet is 1μm.

[0053] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, so that the silver channel and the drug delivery channel at the tip of the dual-channel nanotube are exposed, 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 to form 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, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of organic matter in a single cell.

[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, 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 on the tip surface of the dual-channel nanotube, and an ion-sensitive functional layer is deposited. Two copper conductive metal wires are introduced, one connected to the mercury in the dual-channel glass tube and the other connected to the outer surface of the functional layer. Subsequently, a diamond-like protective layer is deposited by plasma vapor phase.

[0059] S3, refer to Figure 1 As shown in the figure, the atmospheric pressure micro plasma jet mixed with argon and oxygen is used to controllably remove the protective layer at the tip of the probe, exposing a small amount of the functional layer and realizing the fixed-point recording function of specific signals. The flow rate of argon is 40sccm, the flow rate of oxygen is 1sccm, and the diameter of the thinnest part of the jet is 0.5μm.

[0060] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, so that the mercury channel and the drug delivery channel at the tip of the dual-channel nanotube are exposed, 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, thereby realizing the internal reference function. Figure 2 , to obtain implantable nanoprobes, refer to Figure 3 The tip structure of the nanoprobe is, from the inside out, 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, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of ions in a single cell.

[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 is 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 is 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 on the tip surface of the dual-channel nanotube, a molecularly sensitive functional layer is deposited, and two silver conductive metal wires are introduced, one connected to the silver in the dual-channel glass tube and the other connected to the outer surface of the functional layer. Subsequently, a silicon dioxide protective layer is chemically vapor deposited.

[0066] S3, refer to Figure 1 As shown, the atmospheric pressure micro plasma jet of helium and oxygen mixed is used to controllably remove the protective layer at the tip of the probe, exposing a small amount of the functional layer and realizing the fixed-point recording function of specific signals. The flow rate of helium is 20sccm, the flow rate of oxygen is 0.8sccm, and the diameter of the thinnest part of the jet is 1.2μm.

[0067] S4, ref. Figure 1 As shown, a microplasma jet is used to remove the functional layer at the tip of the probe, so that the silver channel and the drug delivery channel at the tip of the dual-channel nanotube 3 are exposed, thereby realizing the drug delivery function. The microplasma jet processing 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 channel containing silver, forming a silver / silver chloride internal reference, thereby realizing the internal reference function. Figure 2 , the prepared implantable nanoprobe was obtained, referring to Figure 3 The tip structure of the nanoprobe is, from the inside out, 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, thereby realizing the functions of in situ internal reference, drug delivery, and reliable point-to-point recording of molecules in a single cell.

[0069] The above-mentioned 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 process the probe tip to expose the surface functional layer, thereby enabling reliable fixed-point recording of specific signals. At the same time, the metal channel and the 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 fixed-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 the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or variations within the scope of the claims, which does not affect the essence of the present invention. The above preferred features can be used in any combination without conflicting with each other.

Claims

1. A method for preparing a nanoprobe integrating in-situ internal reference, drug administration 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 pulling it to form a double-channel nano tip, wherein the double-channel nano tip 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, respectively 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 to achieve fixed-point recording; Use microplasma jet to remove the exposed functional layer tip, so that the metal channel and drug delivery channel of the dual-channel nanotip are exposed to achieve 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 administration and fixed-point recording according to claim 1, characterized in that: The outer diameter of the double-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 double-channel nano tip is 1-1000 nm.

3. The method for preparing a nanoprobe integrating in-situ internal reference, drug administration 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 administration 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 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.

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

6. The method for preparing a nanoprobe integrating in-situ internal reference, drug administration 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 administration 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 the thinnest point is 0.01-1000 μm.

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

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

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