Single-cell NO electrochemical sensor based on fluorinated monomolecular layer modified nanotube electrode and preparation method of single-cell NO electrochemical sensor

Abstract

The invention discloses a single-cell NO electrochemical sensor based on a fluorinated monomolecular layer modified nanotube electrode and a preparation method of the single-cell NO electrochemical sensor, and belongs to the field of electrochemical sensors. The preparation method comprises the following steps: firstly, drawing a borosilicate glass tube into a 200nm tip by a laser drawing instrument, then sputtering a conductive gold layer on the surface of the glass tube by plasma sputtering, insulating the redundant part by PDMS, then manufacturing a hollow nanotube electrode by a connecting wire, and finally self-assembling on the surface of the electrode by a gold-sulfur bond to form a fluorinated monomolecular layer. The nanotube electrode modified with perfluorodecanethiol has good selectivity to NO, can accurately detect NO in a single cell under physiological conditions, and is low in detection limit, short in response time and high in anti-interference capability. According to the invention, the scanning ion conductance microscope and the scanning electrochemical microscope are combined, the cell morphology can be imaged while detection is carried out, and the method is expected to play an important role in the field of biomedical analysis and detection and has commercial potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical sensing detection, and particularly relates to a fluorinated monolayer modified nanotube electrode for detecting the NO release amount in single cells and a preparation method thereof. Background Art

[0002] Generally, the analysis of cell populations gives the average information of a large number of cells; the signals of a very small number of abnormal cells will be masked by the signals of the cell population, leading to misleading conclusions. Therefore, studying at the single-cell level can more accurately and comprehensively understand the cell physiological state and life processes, which is of great significance for the early diagnosis and prevention of cancer. NO is a very typical reactive nitrogen species, which is the main product of intracellular metabolism and also the main source of other nitrogen-containing compounds. Many diseases are related to abnormal NO signals. Due to the high reactivity, fast diffusion rate and short half-life of NO, its in-situ, rapid, quantitative and accurate detection becomes particularly important.

[0003] The methods for NO detection mainly include colorimetry, spectrophotometry, spectroscopy, fluorescence method, and electrochemical method, etc. Among them, the electrochemical method has the advantages of sensitivity, specificity, simplicity, low cost, etc., so an electrochemical sensor can be used to detect NO molecules in real time. In the construction of NO sensors, nano-sized electrodes can be used to pierce into the interior of cells, and the NO release amount of cells can be detected in real time with almost no damage to the cells. The highly fluorinated skeleton is beneficial to the diffusion of NO due to its hydrophobic structure. In this paper, perfluorodecanethiol containing multiple fluorine groups is first applied to the selective detection of NO. They are combined on the gold electrode surface through gold-sulfur bonds and can form regular and uniform fluorinated monolayers, which are suitable for the tip modification of this ultra-microelectrode. Based on this, a good single-cell NO detection system can be constructed.

[0004] The present invention develops an electrochemical sensor capable of directly detecting NO in real time. The preparation method of this sensor is simple and its performance is good, and it is expected to play an important role in the field of biomedical analysis and detection. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorinated monolayer modified nanotube electrode for real-time selective detection of NO in single cells, a preparation method thereof and a detection method.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A single-cell NO electrochemical sensor based on a fluorinated monolayer modified nanotube electrode and a preparation method thereof.

[0008] A nanotube electrochemical sensor for detecting nitric oxide in single cells, which includes a nanotube electrode inserted into a single cell. The nanotube electrode has a single-hole capillary structure. The nanotube is drawn by a puller to form a nanotube with an opening diameter of 200 nm. A conductive Au layer is sputtered on the outer wall of the nanopore by plasma, and a metal wire is connected to the conductive layer. An insulating layer PDMS is coated on the position of the Au layer far from the tip of the nanopore.

[0009] In the nanotube electrode sensor for detecting nitric oxide in single cells, a perfluorodecanethiol monolayer is modified at the position of the Au layer close to the tip of the single-hole nanoporous capillary structure. Through the Au-S bond interaction, perfluorodecanethiol is modified on the Au layer outside the nanopipette. Based on the design of the detection probe, the detection system can selectively detect the change in the intracellular NO concentration.

[0010] By setting five parameters of the laser power, scanning width, shutter speed, delay time, and hard pull strength of the needle puller, the capillary is drawn to obtain a single-hole nanoporous capillary with a preset tip aperture size.

[0011] A gold layer is sputtered on the outer wall of the single-hole nanoporous capillary, and an insulating layer is set at the position of the single-hole nanoporous capillary far from the tip to obtain a single-hole nanoporous capillary structure.

[0012] The nanotube electrode is immersed in an ethanol solution of perfluorodecanethiol and incubated overnight at 4 °C to obtain a nanotube electrode modified with a fluorinated monolayer.

[0013] The mass fraction of perfluorodecanethiol / ethanol in the thiol solution used for the modification varies within the range of 0.05% to 2%.

[0014] The nanotube electrode modified with the fluorinated monolayer is used as the working electrode, and the hollow tube is used as the SICM probe to build a nanotube electrode electrochemical sensor for detecting nitric oxide in single cells.

[0015] A preparation method of a nanotube electrode sensor for detecting nitric oxide in single cells, which includes the steps:

[0016] Using a PBS solution as the electrolyte, the nanotube electrode is inserted into the electrolyte to assemble a NO sensor, and nitric oxide solutions with different concentrations are sequentially added dropwise to the electrolyte. A constant voltage is applied and the I-t curve of the nanotube electrode sensor is measured using an electrochemical workstation.

[0017] A detection method of a modified nanotube electrode electrochemical sensor for detecting nitric oxide in single cells, which includes the steps:

[0018] Detection unit.

[0019] (1) Detection of cell morphology.

[0020] The nanotube electrode is installed on a 3D translation stage, and under microscopic observation, the nanotube electrode is slowly inserted into the single cell to be measured. Using PBS as the electrolyte, a bias voltage V = 0.05 V is applied to the two Ag / AgCl electrodes in the middle of the nanotube electrode and in the bath solution. After obtaining a stable ionic current, the morphology of the single cell is scanned using the jump mode, and the change in the ionic current in the middle of the nanotube electrode is recorded to assist in confirming the process of inserting into the cell.

[0021] (2) Detection of electrochemical signals.

[0022] A voltage V = 0.8 V is applied to the nanotube electrode. After the current stabilizes, dibenzothiophene is used to induce oxidative stress in the single cell to be measured to generate NO, and then the current signal is collected. The NO concentration of the single cell to be measured is obtained based on the collected current signal and the I-t curve.

[0023] The beneficial effects of the technical solution to be protected by the present invention are mainly reflected as follows: Through the design of the detection unit of the nanoelectrode, a fluorinated monolayer with high selectivity for NO detection is modified on the electrode. This method provides good selectivity, has little influence on the size change of the probe, and is more convenient and easier to operate. And the hydrophobic surface of the fluorinated monolayer promotes the aggregation of gaseous NO to the active site, realizing a higher electrochemical response, which is more conducive to information output and accurate judgment. Using a laser pulling pipette, the tip of the capillary can reach the nanoscale, so that the tip can be inserted into the cell for detection without destroying the physiological activity of the cell, and the signal detection of different regions below the cell level such as the cell nucleus and cytoplasm can be realized.

[0024] The technical solution of the present invention combines the scanning ion conductance microscopy and scanning electrochemical microscopy techniques to realize a detection method and test system for the simultaneous output of the nanoelectrode probe unit for cell morphology, cell localization, and intracellular NO electrochemical signals. And

[0025] The present invention can effectively ensure non-destructive detection, realize in-situ detection of signals in living cells, and achieve the timeliness of detection signals, which is of great significance for the early screening of diseases and clinical medical diagnosis. The cell activity is not damaged, and in-situ detection of intracellular target signals is realized, improving the effectiveness and timeliness of cell signal output.

[0026] The present invention provides an electrochemical sensor based on a modified nanotube electrode for detecting nitric oxide in single cells. Based on the nanotube electrode, by virtue of the highly hydrophobic property of the fluorine skeleton, selective detection of NO is carried out. The modified nanotube electrode with a fluorinated self-assembled monolayer is used to achieve sensitive detection of nitric oxide in single cells, and the detection limit can reach 1.8 nM. The structure of the present invention is simple, the device size is small, and the interference to single cells is small when inserted into single cells, hardly affecting the normal physiological activities of cells. Therefore, in-situ and dynamic monitoring of nitric oxide release can be realized. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0028] Figure 1 It is a structural diagram of the use state of a fluorinated monolayer modified nanotube electrode sensor for detecting nitric oxide in single cells.

[0029] Figure 2 It is a scanning electron microscope image of the tip of the nanotube electrode in the fluorinated monolayer modified nanotube electrode sensor for detecting nitric oxide in single cells of the present invention. (a) Side view and (b) top view of the nanorod electrode before insulation; (c) side view and (d) top view of the nanorod electrode after insulation.

[0030] Figure 3 It is the contact angle measurement results before (left) and after (right) modifying the fluorinated monolayer interface in the fluorinated monolayer modified nanotube electrode sensor for detecting nitric oxide in single cells of the present invention.

[0031] Figure 4 It is a flowchart of the preparation method of a fluorinated monolayer modified nanotube electrode sensor for detecting nitric oxide in single cells according to the present invention.

[0032] Figure 5 It is the cyclic voltammogram of the nanotube electrode (a) before insulation and (b) after insulation in a ferrocene methanol solution.

[0033] Figure 6 (a) is the I-t curve obtained by testing the fluorinated monolayer modified nanotube electrode in PBS containing different concentrations of nitric oxide, Figure 6 (b) is the corresponding linear relationship diagram.

[0034] Figure 7 It is the result diagram of the selective test of the prepared modified nanotube electrode for different interfering components.

[0035] Figure 8Ion current change diagram obtained during the process of inserting into cells. Among them, (a) bright-field microscope image before inserting into cells; (b) above the cell; (c) inserting into cells; (d) removing from cells; (e) ion current during the approach and penetration of HepG2 cells.

[0036] Figure 9 Scanned 2D (left) and 3D (right) morphology diagrams of HepG2 cells.

[0037] Figure 10 NO oxidation current measured after adding different concentrations of dibenzothiophene stimulation after the electrode is inserted into the cell interior. Specific implementation manners

[0038] The following illustrates the implementation manners of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. Based on the implementation manners in the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present invention.

[0039] Please refer to Figure 1 , the present invention provides a fluorinated monolayer modified nanotube electrode for detecting nitric oxide in single cells. As shown in the figure, it includes a modified nanotube electrode inserted into a single cell. A gold layer is sputtered on the outer wall of the nanotube electrode structure, and an insulating layer is provided at a position away from the tip of the nano single-hole capillary structure: a fluorinated monolayer is provided at a position of the gold layer close to the tip of the nanotube structure.

[0040] The modified nanotube electrode provided by the present invention belongs to a novel high-sensitivity biosensor. The present invention first realizes the detection of nitric oxide in single cells with the help of a fluorinated monolayer. With the help of the hydrophobic structure of PFDT, the tiny current signal of NO released by a single cell can be selectively detected, which has higher detection sensitivity than traditional electrochemical nanoelectrode sensing technology, and the detection limit can reach 1.8 nM. The successful application of the fluorinated monolayer modified nanotube electrode to the detection of nitric oxide in single cells provides a new idea for high-sensitivity detection in the future fields of single-cell analysis and biosensing, and is also applicable to the detection of other chemical components in single cells, with a very broad application scenario.

[0041] In some implementation manners, the nano single-hole capillary is selected from borosilicate capillaries.

[0042] In some embodiments, a method for preparing a fluorinated monolayer-modified nanotube electrode for detecting nitric oxide in a single cell is also provided, as Figure 4 shown, which includes the steps:

[0043] Preparation of a nanopipette electrode.

[0044] The used borosilicate glass tube (BF100-58-10) with a length of 10 cm, an outer diameter of 1 mm and an inner diameter of 0.58 mm and the used laser puller (P-2000, Sutter Instruments, https: / / www.sutter.com / MICROPIPETTE / p-2000.html) are both from Sutter Instruments Company in the United States. By adjusting the parameters of the laser puller, five parameters including the laser power (HEAT), the scanning width (FIL), the shutter speed (VEL), the delay time (DEL), and the hard pull strength (PUL), using the program: Line1: HEAT = 370; FIL = 3; VEL = 30; DEL = 200;; PUL = 0; Line2: HEAT = 370; FIL = 3; VEL = 40; DEL = 180;; PUL = 250, a nanopipette with a tip diameter of 200 nm is obtained.

[0045] Using a plasma sputtering instrument, set the pressure to 20 Pa and the current to 12 mA, and sputter a gold layer on the outer wall of the nanopore capillary to form a conductive layer.

[0046] The preparation method of the fluorinated monolayer-modified nanotube electrode sensor of the present invention will be described below through specific examples:

[0047] Wind a metal wire connected to the conductive layer around the outer wall of the nanopore capillary, and dip in PDMS diluted with tert-butanol at a position far from the tip of the nanotube electrode, where the volume ratio of PDMS to tert-butanol is 1:6. Use this solution to set the insulating layer.

[0048] After the nanotube electrode is insulated, expose a fixed electrode area and modify the fluorinated monolayer. Dissolve perfluorodecanethiol in ethanol to make the mass fraction of perfluorodecanethiol in the resulting solution 0.1 wt%, incubate the prepared nanotube electrode with this mixed solution and store it overnight in the dark at 4°C. It needs to be purged with nitrogen to remove oxygen before use. After the modification is completed, take out the electrode, rinse it with ethanol, and dry it in the air.

[0049] Perform performance tests on the nanotube electrode prepared by the present invention, and the results are as Figure 3 and Figure 5 shown, Figure 5The cyclic voltammogram of the nanotube electrode in a ferrocene methanol solution in a traditional electrochemical test shows that the current is about 1.2nA, which can be used to detect weak changes in intracellular biomolecules. Figure 3 The change in contact angle of the electrode before and after modification with the fluorinated monolayer is shown, and a significant increase in hydrophobicity can be observed, indicating the successful assembly of the monolayer.

[0050] In some embodiments, a method for detecting nitric oxide in a single cell using a nanotube electrode sensor is provided, comprising the steps of:

[0051] Using PBS solution as the electrolyte, the fluorinated monolayer-modified nanotube electrode was assembled together with an electrochemical workstation, a counter electrode (platinum wire electrode), and a reference electrode (Ag / AgCl electrode) to form a NO electrochemical sensor.

[0052] The detection method of the fluorinated monolayer modified nanotube electrode NO sensor of the present invention is further explained below through specific examples:

[0053] Detection of nitric oxide content in electrolyte: A three-electrode system was composed of 10 mM PBS as electrolyte, Ag / AgCl as reference electrode, Pt wire as counter electrode, and modified nanotube electrode as working electrode. Different concentrations of nitric oxide were added to the solution in turn for measurement. The constant voltage V=0.8 V was set, and the chronovoltammetric curve was obtained using the electrochemical workstation CHI920d.

[0054] In this embodiment, Figure 6 The It curve is obtained by testing the fluorinated monolayer modified nanotube electrode in PBS containing different concentrations of nitric oxide. It can be seen from the figure that as the NO concentration increases, the current I obtained shows a good step-up change with the concentration. It can be seen from the working curve that the obtained linear relationship is good. This result shows that the detection limit of the fluorinated monolayer modified nanotube electrode of the present invention for NO is 1.8nM, and a good linear relationship is shown between 1.8nM-6.4μM and 6.4μM-78.9μM.

[0055] Cultivation of HepG2 cells.

[0056] HepG2 cells were cultured on a cell culture dish. HepG2 cells were cultured in a complete DMEM medium consisting of 45 ml DMEM medium, 5 ml fetal bovine serum (FBS) and 0.5 ml double antibody, and the cells were cultured in a 37° C. constant temperature incubator containing 5% CO 2 and 95% air.

[0057] SICM Characterization of HepG2 Cells: During the SICM scanning process, the culture dish was mounted on the substrate. The modified nanotube electrode was mounted on a 3D manipulator. The position near a single cell was located under the assistance of an inverted optical microscope. The SICM scanning was performed in a hopping mode. As the distance between the capillary and the scanned sample decreased, the ionic current flowing through the two Ag / AgCl electrodes between the bath solution and the nanotube decreased.

[0058] The detection change limit was set to 3% of the current, and the probe moving speed was set to 10 μm / s.

[0059] Determination of the approach curve.

[0060] As Figure 1 shown in the left half. Two Ag / AgCl electrodes were used, one located inside the nanotube electrode and the other in the bulk solution. A stable ionic current could be obtained when a bias voltage was applied between these two electrodes. When the distance between the nanotube tip and the cell decreased to a very small value, the ionic current decreased due to steric hindrance. At this time, a graph was plotted with the current change as the Y-axis and the probe travel distance as the X-axis to obtain the approach curve. This method could determine the position of a single cell to be measured, which was used for scanning the cell morphology and determining the process of the probe piercing into the cell. The process was as Figure 8 shown.

[0061] The nanotube electrode was fixed on a 3D manipulator, and the scanned cell morphology was as Figure 9 shown.

[0062] Detection of Nitric Oxide in HepG2 Cells.

[0063] The relative position of the probe movement could be calculated through the obtained 2D image, so that the probe could be moved to the position point where it was needed to pierce into the cell for detection. Under the microscope observation, the fluorinated monolayer modified nanotube electrode was inserted into the single cell to be measured.

[0064] A constant voltage of +0.8 V was applied to the electrode. After the current was stable, different concentrations of dibenzothiophene were continuously dropped into the culture dish to induce the production of nitric oxide by oxidative stress in the single cell to be measured and then collect its oxidation signal as Figure 10 , and the nitric oxide release amount of the single cell to be measured was obtained according to the measured current signal and the chronoamperometric curve.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of a single-cell NO electrochemical sensor based on a fluorinated monolayer modified nanotube electrode, characterized in that, Including the following steps: Using a laser pipette puller to draw a borosilicate glass tube to obtain an open nano - pipette; Placing the nano - pipette drawn by the pipette puller into the sputtering chamber of a plasma sputtering instrument, adjusting the pressure to 20 Pa, adjusting the sputtering current to 12 mA, and sputtering both the front and back sides of the nano - pipette for 5 minutes each to obtain a gold layer with a thickness of 40 - 50 nm; Partially insulating the electrode by dipping the gold nano - electrode into a PDMS solution to expose a fixed electrode area; Immersing the gold nano - electrode in a perfluorodecanethiol solution and incubating it overnight at 4 °C in the dark; through the gold - sulfur bond interaction, perfluorodecanethiol is modified on the surface of the gold layer of the nano - pipette to obtain a gold nano - electrode modified with a fluorinated monolayer; Using the gold nano - electrode modified with a fluorinated monolayer as a probe, combining scanning ion conductance microscopy and scanning electrochemical microscopy techniques: (1) Scanning cell morphology and locating cell position For cell morphology detection, an electrochemical workstation and a two - electrode system are used. A circuit is formed through an Ag / AgCl electrode placed in the middle of the nanotube electrode and an Ag / AgCl electrode placed in the electrolyte to generate an ionic current. Morphology imaging is generated through the obstruction of the ionic current by the cell morphology; through the electrochemical workstation, the processed nano - electrode is fixed on a holder; using the Ag / AgCl electrode inserted into the middle of the nano - pipette as the working electrode, and another Ag / AgCl electrode placed in the PBS solution as the reference electrode, applying a voltage of +0.05 V; Continuously approaching the nano - pipette to the cell to be measured through a micromanipulation system, and recording the current change at the tip of the nano - pipette; continuously obtaining the current change within a 50 μm * 50 μm area through the nano - pipette to scan and obtain the morphology image of the cell; (2) Detection of intracellular electrochemical signals Based on the image obtained by the electrochemical workstation and morphology detection, calculating the moving position of the nano - electrode to insert into the cell interior at a specified position; for the detection of electrochemical signals after inserting into the cell, an electrochemical workstation and a three - electrode system are used. The modified fluorinated monolayer selectively allows NO to pass through and is oxidized on the surface of the nanotube electrode to generate corresponding electrochemical signals; using the gold nanotube electrode as the working electrode, Ag / AgCl as the reference electrode, and a platinum wire electrode as the counter electrode, applying a voltage of +0.8 V, and collecting the current signal with the electrochemical workstation to obtain the current signal of NO inside the cell.

2. The method according to claim 1, wherein The opening diameter of the nano - pipette is 180 - 200 nm.

3. The method according to claim 1, wherein The PDMS solution is prepared by mixing PDMS and tert - butanol at a volume ratio of 1:5 - 1:

8.

4. The preparation method of a single-cell NO electrochemical sensor based on a fluorinated monolayer-modified nanotube electrode according to claim 1, wherein, The perfluorodecanethiol solution is a solution with nitrogen purged to remove oxygen, using ethanol as the solvent, and the mass fraction of perfluorodecanethiol is 0.01% - 1%.

5. An NO electrochemical analysis system prepared by using any of the methods described in claims 1 - 4.

6. The analysis system according to claim 5, for detecting the NO release amount in a single cell.

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