Method for real-time analysis of biomolecules in single living cell by high spatial resolution liquid-modified nano-electrode and application

Abstract

The application provides a method for real-time analysis of biomolecules in single living cells by high spatial resolution liquid modified nanoelectrode, which establishes a nanoelectrode for single cell electroanalysis by filling kit components into nanotubes. After inserting the nanoelectrode into living cells, the kit components are injected into the cells by electrochemical method, so as to react with target biomolecules. The by-product hydrogen peroxide is electrochemically detected by the annular electrode at the tip of the nanoelectrode, so as to complete the quantification of the target biomolecules in the whole cell. The kit components are retained in the solution, which avoids the complexity of electrode modification and probe design, so that more intracellular biomolecules can be detected.

Description

Technical Field

[0001] This application relates to the field of electrochemical analysis, specifically to a method and application for real-time analysis of biomolecules in a single living cell using a high spatial resolution liquid-phase modified nanoelectrode. Background Technology

[0002] Single-cell analysis allows for precise quantification of heterogeneity among individual cells, crucial for detecting trace biomolecules at the subcellular level. Recent studies have revealed subcellular heterogeneity, particularly in the subcellular localization of proteins and associated miRNAs. New subcellular information can provide clues for studying biochemical mechanisms. Therefore, real-time spatial analysis of biomolecules within single living cells at the subcellular level is essential. In recent research, super-resolution fluorescence imaging has overcome the diffraction limit, enabling visualization of subcellular structures and biomolecules within single cells. However, this technique heavily relies on the design of strong emission and highly specific fluorescent probes / labels, which are not easily achieved. Furthermore, factors such as fluorescence stability, cell damage, and cytotoxicity must be considered, further complicating the acquisition of satisfactory probes. Therefore, super-resolution fluorescence microscopy cannot be simply applied to the real-time measurement of various biomolecules at the subcellular level within single living cells.

[0003] Chemically modified nanoelectrodes are another tool for analyzing biomolecules in single living cells. Due to their extremely small size, nanoelectrodes can penetrate single living cells without significantly disrupting cellular activity. When measuring electroactive molecules (such as reactive oxygen species and neurotransmitters), changes in current are recorded by utilizing the direct electron transfer of the target molecule on the electrode. The rapid molecular diffusion and subsequent electron transfer of nanoelectrodes give them high detection sensitivity and spatiotemporal resolution. To analyze more non-electroactive biomolecules, enzymes or recognition elements (such as glucose and cholesterol) are modified on the electrode to assemble a solid-phase functional layer. Although such modified electrodes have achieved many successes in single-cell electrochemical analysis, this type of solid-phase modified sensor still faces some problems: (1) the limited number of enzymes or recognition elements modified on the electrode surface restricts the types of biomolecules that can be analyzed within the cell; (2) enzymes immobilized on the electrode surface lose some activity compared to enzymes in the liquid phase, their movement speed is relatively slow, and the detection sensitivity is low. Summary of the Invention

[0004] To address the current limitations of subcellular level detection, this application provides a method for real-time analysis of biomolecules within a single living cell using a high spatial resolution liquid-modified nanoelectrode, characterized by the following steps:

[0005] Quartz nanocapillaries were drawn using a laser needle drawing instrument to obtain open nanotubes;

[0006] Using methane as the carbon source and argon as the protective agent, a layer of carbon was deposited on the inner surface of a quartz nanocapillary by chemical vapor deposition at 900℃ to 1000℃ to obtain a carbon nanoelectrode.

[0007] In a solution containing chloroplatinic acid, the deposited carbon nanoelectrode was activated by scanning potentials from 0 mV to −500 mV, and a gradual increase in reduction current was observed.

[0008] Platinum black deposition on the carbon wall is completed through a constant potential step from -50 mV to -100 mV before the current increases sharply;

[0009] Platinum-plated carbon nanoelectrodes are immersed in a solution containing reagent kit components. A negative pressure is applied to separate droplets of the reagent kit components into the nanoelectrodes, enabling the detection of intracellular biomolecules. Specifically, hydrogen peroxide is generated by the reaction of the reagent kit components with the target biomolecules in the cells, thus completing the quantitative detection of biomolecules throughout the cell.

[0010] This application also provides an application of the analytical method described herein in single-cell detection. Beneficial effects

[0011] This application establishes a nanoelectrode for single-cell electroanalysis by filling kit components into nanotubes. After the nanoelectrode is inserted into a living cell, the kit components at the tip react with the target biomolecules within the cell. The generated byproduct, hydrogen peroxide, is electrochemically detected by a ring electrode at the nanoelectrode tip, thus quantifying the target biomolecules throughout the cell. The kit components remain in solution, avoiding the complexities of electrode modification and probe design, and addressing the problem of insufficient solid-phase modification sites, thereby enabling the detection of more intracellular biomolecules.

[0012] Further advancements in subcellular detection involve the electrochemical extraction of specific cellular vesicles or organelles into nanotubes, with the reagent reaction occurring at the tip of the nanoelectrode for electrochemical analysis of biomolecules. While single-cell nanoelectrode methods can facilitate electrochemical analysis and detect more intracellular biomolecules at the subcellular level, the sorting process and subsequent reagent reaction hinder real-time analysis. Therefore, innovations in nanoelectrode design are needed to continuously achieve these goals. This application designs a liquid-phase modified nanoelectrode for real-time and localized detection of biomolecules within a single living cell to collect spatial information. Unlike solid-phase modification of electrodes with chemicals, a nanodroplet containing all detection reagents is retained at the tip of a platinum-plated hollow carbon nanoelectrode (Pt@CNP).

[0013] The nanoelectrode has a very narrow pore, resulting in extremely slow liquid diffusion. Consequently, the droplet remains stably retained at the tip, forming a solid-phase modified layer. No modification process is involved, therefore any reagent can be used for detection. Depending on the nanoelectrode's position within the cell, reagents at the nanoelectrode pore can react with target biomolecules to generate hydrogen peroxide for electrochemical measurement. Because the reagents remain in the liquid phase, they maintain high reactivity to maximize conversion efficiency. Therefore, this liquid-phase modified nanoelectrode not only retains the high spatiotemporal resolution advantage of solid-phase modified nanoelectrodes in single-cell analysis but also exhibits high detection capability for low-abundance biomolecules at the subcellular level. Ultimately, the spatial distribution of biomolecules within a single living cell is obtained, enabling real-time subcellular analysis. Attached Figure Description

[0014] Figure 1 A schematic diagram of the detection principle in a single cell according to an embodiment of this application;

[0015] Figure 2 An embodiment of this application describes the electrochemical measurement of a liquid-phase modified nanoelectrode; wherein (A) the current of a nanoelectrode containing glucose oxidase is recorded; (B) the current response of different glucose concentrations in A is statistically analyzed; (C) the currents of sphingomyelin, alkaline phosphatase, and choline oxidase in the nanoelectrode are recorded; and (D) the current response of SMase in C at different activities is statistically analyzed.

[0016] Figure 3 In one embodiment of this application, glucose analysis of a single live MCF-10A cell is performed; wherein (A) a bright-field image of a 70 nm diameter nanoelectrode penetrating a single live cell; (B) current records collected by the nanoelectrode before and after the glucose oxidase penetrates the cell by the nanodroplet; (C) statistical current response of a single 25 cells; and (D) fluorescence image of a nanoelectrode loaded with 500 μM fluorescein positioned in a single live cell for 5 minutes.

[0017] Figure 4 In one embodiment of this application, SMase activity analysis of a single CT26 cell was performed; (A) Current records of sphingomyelin, alkaline phosphatase, and choline oxidase collected by a nanoelectrode before and after nanodroplets penetrated into a single live CT26 cell; (B) Current response of SMase activity collected in a single CT26 cell (n=5); (C) Bright-field images of different regions (ae) of a single CT26 cell penetrated by a nanoelectrode; (D) Current records collected by a nanoelectrode before and after penetration into a single CT26 cell. ae represents five locations from cell c; (E) Statistical analysis of pericellular and nuclear currents (n=4);

[0018] Figure 5(A) Preparation process of liquid-phase modified nanoelectrode; (B) TEM image of nanoelectrode after platinumization;

[0019] Figure 6 Cyclic voltammogram of a liquid-phase nano-modified electrode in one embodiment of this application;

[0020] Figure 7 In one embodiment of this application, a platinum-plated carbon nanotube electrode is used without glucose oxidase as a control experiment.

[0021] Figure 8 Schematic diagram of the sphingomyelinase (SMase) reaction mechanism;

[0022] Figure 9 A schematic diagram of a control experiment using nanoelectrodes in nanodroplets without adding glucose oxidase in one embodiment of this application;

[0023] Figure 10 Cell fluorescence image after electrochemical pumping of fluorescein (500 μM) from a nanopipette in one embodiment of this application. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0025] In one embodiment, a method for real-time analysis of biomolecules within a single living cell using a high spatial resolution liquid-phase modified nanoelectrode is provided, comprising the following steps:

[0026] Quartz nanocapillaries were drawn using a laser needle drawing instrument to obtain open nanotubes;

[0027] Using methane as the carbon source and argon as the protective agent, a layer of carbon was deposited on the inner surface of a quartz nanocapillary by chemical vapor deposition at 900℃ to 1000℃ to obtain a carbon nanoelectrode.

[0028] In a solution containing chloroplatinic acid, the deposited carbon nanoelectrode was activated by scanning potentials from 0 mV to −500 mV, and a gradual increase in reduction current was observed.

[0029] As the current increases to -10 pA to -50 pA, platinum black deposition on the carbon wall is completed through a potentiostatic step between -50 mV and -100 mV.

[0030] Platinum-plated carbon nanoelectrodes are immersed in a solution containing reagent kit components. A negative pressure is applied to separate droplets of the reagent kit components into the nanoelectrodes, enabling the detection of intracellular biomolecules. Specifically, hydrogen peroxide is generated by the reaction of the reagent kit components with the target biomolecules in the cells, thus completing the quantitative detection of biomolecules throughout the cell.

[0031] In one embodiment, the opening diameter of the nanotube is 50nm-80nm.

[0032] In one embodiment, the concentration of the chloroplatinic acid is from 1 mM to 5 mM.

[0033] In one embodiment, the thickness of the deposited carbon layer is 5 nm to 10 nm.

[0034] In one embodiment, the ratio of methane to argon is 5:3.

[0035] In one embodiment, the applied negative pressure is 0.5 to 1 bar, and the negative pressure duration is 5 to 15 minutes.

[0036] In one embodiment, the intracellular biomolecule to be detected is glucose, and the kit component is glucose oxidase. An enzyme solution of 5 to 20 mg / mL glucose oxidase is sorted into a nanoelectrode to obtain a liquid-phase glucose oxidase-modified nanoelectrode.

[0037] In one embodiment, the intracellular biomolecule to be detected is sphingomyelin oxidase. The kit components are sphingomyelin, alkaline phosphatase, and choline oxidase. A mixture containing 1 to 2 mM sphingomyelin, 2 to 6 U / mL alkaline phosphatase, and 2 to 6 U / mL choline oxidase is sorted into a nanoelectrode to obtain a liquid-phase modified nanoelectrode containing nanodroplets of the sphingomyelin oxidase kit components.

[0038] One embodiment provides the application of the described analysis method in single-cell detection.

[0039] In one embodiment, quartz capillaries (Q100-50-7.5) were drawn using a laser needle puller (P-2000, Sutter Instruments) to obtain nanotubes with an opening diameter of 70 nm. Using methane as the carbon source and argon as the protective agent (methane / argon: 5 / 3), a carbon layer with a thickness of 5-10 nm was deposited on the inner surface of the quartz nanocapillaries (CNPs) at 950 °C via chemical vapor deposition (CVD). Then, the CNP nanoelectrode was activated in a solution containing 4 mM chloroplatinic acid by scanning potentials from 0 to −500 mV, and a gradual increase in reduction current was observed. Subsequently, platinum black deposition on the carbon walls was completed via a constant potential step of −80 mV before a sharp increase in current. Finally, the platinum-plated carbon nanoelectrode was immersed in a solution containing the kit components, and a negative pressure of 0.5 to 1 bar was applied for 5 to 10 minutes to sort droplets onto the nanoelectrode, thus creating a liquid-phase modified nanoelectrode. For glucose detection, an enzyme solution of 5 to 20 mg / mL glucose oxidase is sorted into a nanoelectrode to obtain a liquid-phase glucose oxidase-modified nanoelectrode. For sphingomyelin oxidase detection, a mixture containing 1 to 2 mM sphingomyelin, 2 to 6 U / mL alkaline phosphatase (ALP), and 2 to 6 U / mL choline oxidase is sorted into a nanoelectrode to obtain a liquid-phase modified nanoelectrode containing nanodroplets of sphingomyelin oxidase kit components. See the schematic diagram of the detection principle. Figure 5 .

[0040] Electrochemical response of liquid-phase modified nanoelectrodes

[0041] By transmission electron microscopy (TEM, see...) Figure 5 The diameter of the tip pore was characterized as approximately 70 nm. To estimate the volume of the tip nanodroplet, 10 mm of phosphate-buffered saline (PBS, pH 7.4) and 100 mm of K₄[Fe(CN)₆] were loaded into the nanodroplet. Cyclic voltammetry showed an oxidation charge of 1.2 pC (see [link to citation]). Figure 6 The volume of the nanodroplets is estimated according to Formula 1.

[0042] V = Q / cnF (eq 1)

[0043] In the formula, c is the concentration of K₄[Fe(CN)₆] in the nanodroplet (100 mM), n is the number of transferred electrons (n=1), and F is the Faraday constant (96485 c / mol). Based on this, the volume of the nanodroplet is calculated to be 0.12 fL, and the length is approximately 1 μm.

[0044] Detection of glucose and sphingomyelinase using liquid-phase modified nanoelectrodes

[0045] The electrochemical performance of liquid-phase modified nanoelectrodes was evaluated using glucose and sphingomyelinase (SMase) as two important biomolecules. For glucose detection, glucose oxidase was added to the nanodroplets to oxidize glucose into hydrogen peroxide for electrochemical quantification. Similar to the characterization of solid-phase modified nanoelectrodes, chronoamperometry experiments were performed on liquid-phase modified nanoelectrodes with different glucose concentrations at 0.6 V. After a stable background current was collected, a significant increase in current was immediately observed upon the addition of 50 μM glucose. Figure 2 (A). Further addition of glucose to the solution resulted in a linear increase in the current response. Figure 2 (B) The control experiment used a platinum-plated carbon nanotube electrode without glucose oxidase loading. No increase in current was observed before or after the addition of glucose. Figure 7 ). Continuous observation of the reaction over 30 minutes revealed steady-state characteristics. Figure 2 As shown in Figure A, the rapid and stable oxidation of glucose by glucose oxidase in a droplet at the needle tip is demonstrated. Its excellent responsiveness and stability confirm that glucose oxidase in the droplet can serve as a stable enzyme layer for glucose determination. More importantly, the liquid-phase modified nanoelectrode exhibits a sensitivity of 0.15 pA mM. -1 nm -1 The sensitivity is significantly higher than that of the glucose oxidase-modified nanoelectrode (0.01 pA mM). -1 nm -1 The increased sensitivity confirmed the high turnover rate of the enzyme in the liquid state.

[0046] Liquid-modified nanoelectrodes not only possess high detection sensitivity but also enable the detection of complex biomolecules that are difficult to detect using optical and electrochemical sensors. To demonstrate this property, this application selects SMase as the demonstration model. The determination of SMase requires complex reactions (such as...). Figure 8 As shown, the process includes SMase cleaving sphingomyelin to generate phosphocholine and ceramide, phosphocholine reacting with alkaline phosphatase to generate choline, and choline oxidase oxidizing it to generate hydrogen peroxide for detection. All these components are not easily assembled on nanoelectrodes, but can be mixed in nanodroplets.

[0047] See this application Figure 2 Electrochemical measurements of liquid-phase modified nanoelectrodes. Figure 2 (A) Current was recorded using a nanoelectrode containing glucose oxidase to analyze glucose concentrations ranging from 50 to 1000 μM. Inset: Current response of 1 mm glucose over 30 min was continuously recorded. Figure 2 (B) Statistical analysis of the current response of different glucose concentrations in A. Figure 2In the middle (C), the currents of sphingomyelin, alkaline phosphatase and choline oxidase in the nanoelectrode were recorded, and the different activities of SMase in the range of 0.025 ~ 2u / mL were analyzed. Figure 2 The current response of SMase C at different activities is statistically analyzed in (D). The red line represents the linear fitting curve, and the error bars represent the standard deviation of five different nanoelectrodes. The buffer was 10 mM PBS, and the applied voltage was 0.6 V vs. Ag / AgCl.

[0048] When a nanoelectrode containing droplets is immersed in a solution containing SMase, a steady-state current response is obtained with a response time of 0.1 s. This direct reaction demonstrates the rapid diffusion and transformation of SMase, and the generated hydrogen peroxide is similar to that produced by a solid-phase modified sensor. Figure 2 (C). The response is linearly related to SMase activity. Figure 2 The sensitivity was 0.14 pA (U / mL). -1 nm -1 This is superior to the previously developed nano-kit-based electrochemical assay (0.02 pA (U / mL)). -1 nm -1 The results showed that the reaction of SMase with the kit components at the nanoelectrode pores generated hydrogen peroxide near the Pt layer, thereby promoting the complete consumption of hydrogen peroxide on the electrode. Therefore, the liquid-modified nanoelectrode exhibited good detection sensitivity.

[0049] Single live cell biomolecular analysis

[0050] The established liquid-phase modified nanoelectrode was inserted into a single live cell for glucose and SMase analysis. Baseline currents were recorded by the extracellular liquid-phase glucose oxidase-modified nanoelectrode. The nanoelectrode was then inserted into a single live MCF-10A cell. Figure 3 In the middle (A), a steady current increase of ~2 Pa was observed. When the electrode was removed from the cell, the current immediately dropped back to the initial background value (A). Figure 3 (See section B). The current response was only observed when the nanoparticles were placed inside cells, indicating that intracellular glucose reacts with glucose oxidase within the microdroplets, generating an electrochemical response. This application also demonstrates a control experiment using nanoelectrodes in nanodroplets without the addition of glucose oxidase, showing no increase in current after insertion into cells. (See section B). Figure 9 This result indicates that the influence of other intracellular biomolecules on the current is negligible, demonstrating the good specificity of liquid-phase enzyme-modified nanotubes. Analysis of 25 MCF-10A cells using nanotubes showed the following current response: Figure 3As shown in Figure C, the mean reaction value was 4.47 ± 4.44 pA, with a relative standard deviation of 99.3%, indicating high heterogeneity in intracellular glucose levels. Based on the calibration curve acquired in solution, the intracellular glucose level was estimated to be 0.6 mM.

[0051] Although nanoelectrodes can anchor nanodroplets at their tips, some diffusion of the chemical substances is unavoidable. To investigate the extent of intracellular diffusion, the fluorescent dye fluorescein was added to the droplets while the nanoelectrodes were placed inside cells. Within 5 minutes, even with a voltage of 0.6 V applied to the carbon layer of the nanoelectrode, only a small fluorescent spot (~0.5 μm) was observed inside the cell. Figure 3 (Middle D). This size is close to the display limit of the fluorescence microscope of this application, indicating that no liquid leakage occurred from the nanodroplets. For comparison, a small voltage of 0.6 V was applied to the silver wire inside the nanoelectrode to initiate electrochemical pumping. Clear fluorescence was visible throughout the cell within 3 s, indicating that liquid flowed out of the pipette (see [reference]). Figure 10 Therefore, compared with existing nanoelectrodes for analyzing biomolecules within a single cell, the established liquid-phase modified nanoelectrodes can perform local measurements, providing spatial and real-time information within the cell.

[0052] Components such as sphingomyelin, alkaline phosphatase, and choline oxidase were incorporated into nanodroplets, and the activity of SMase in single CT26 cells was analyzed using liquid-phase modified nanodroplets. CT26 cells are an undifferentiated colon cancer cell line that exhibits high expression / activity of basic SMase in the cytoplasm and does not require any ion stimulation (such as Mg2+). Similar to single-cell glucose analysis, after inserting nanoelectrodes into the cells, a current response of 1.5 pA could be collected. Figure 4 (A). Removal of the nanoelectrode from the battery caused the current to drop to baseline. Removal of the kit component from the nanodroplet within the nanoelectrode resulted in the disappearance of the current response. All these results confirm the native activity assay of SMase in single live cells. Analysis of five single CT26 cells showed a mean current response of 0.76 ± 0.32 pA. Figure 4 The relative standard deviation was 42%. Based on these current responses, the activity of SMase in a single CT26 cell was estimated to be 0.08 U / mL. The time to reach a steady-state response in a single cell was less than 2 s, as fast as that of a solid-phase modified nanoelectrode. Therefore, the liquid-phase modified nanoelectrode exhibits the ability to perform real-time measurements in single-cell analysis. This is the first time that a nanoelectrode has been used to detect enzyme activity in a single live cell in real time.

[0053] Spatial analysis of SMase activity in CT26 cells.

[0054] Because nanoelectrodes have nanometer-sized openings, they should be able to perform highly spatial measurements of intracellular biomolecules, thus providing spatial information. Experimentally, nanoelectrodes were placed in five regions of a cell, such as... Figure 4 As shown in Figure C, different current responses were observed from these regions using the same nanoelectrodes, revealing a significant heterogeneity in SMase activity within individual cells. Figure 4 (D). Analysis of four single cells showed that the response near the nucleus was almost twice that collected from the periphery of the cell. Figure 4 (E). Simultaneously, the reaction values ​​in the central region of the cell were similar to those in the periphery, indicating almost identical enzyme activity. It is well known that sphingomyelin is an important component of chromatin and the nuclear membrane, and it acts as a second messenger for the production of ceramides under the action of SMase. Therefore, the activity of SMase around the cell nucleus may be higher than that in other cellular regions. This finding supports the idea that nanoelectrodes can provide space and real-time observation for the study of subcellular activity, which is impossible with solid-phase modified nanoelectrodes. Thus, the transformation from solid-phase to liquid-phase modification design has enhanced the detection capabilities of nanoelectrochemical analysis, greatly promoting the development of single-cell electrochemical analysis.

[0055] The above are merely preferred embodiments of the present invention. It should be noted that, for those skilled in the art, numerous improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for real-time analysis of biomolecules in a single living cell by high spatial resolution liquid modified nanoelectrode, characterized in that, The method comprises the following steps: drawing a quartz nanocapillary by using a laser needle drawing instrument to obtain an open nanotube; depositing a carbon layer on the inner surface of the quartz nanocapillary by chemical vapor deposition at 900-1000 ℃ with methane as a carbon source and argon as a protective agent to obtain a carbon nanoelectrode; activating the carbon nanoelectrode by scanning the potential from 0 mV to -500 mV in a solution containing chloroplatinic acid, and observing that the reduction current gradually increases; when the current increases to -10 pA to -50 pA, the platinum black deposition on the carbon wall is completed by a constant potential step between -50 mV and -100 mV; immersing the platinum-coated carbon nanoelectrode in a solution containing reagent kit components, applying negative pressure, and sorting a droplet of the reagent kit components into the nanoelectrode to detect biomolecules in cells, wherein the quantitative detection of biomolecules in cells is completed by the reaction of the reagent kit components with the biomolecules to be detected in the cells to generate hydrogen peroxide; the open diameter of the nanotube is 50-80 nm.

2. The method of claim 1, wherein, the concentration of the chloroplatinic acid is 1-5 mM.

3. The method of claim 1, wherein, the thickness of the deposited carbon layer is 5-10 nm.

4. The method of claim 1, wherein, the ratio of methane to argon is 5:

3.

5. The method of claim 1, wherein, the pressure applied by the negative pressure is 0.5-1 bar, and the negative pressure time is 5-15 minutes.

6. The method of claim 1, wherein, the biomolecule to be detected in the cells is glucose, the reagent kit component is glucose oxidase, and 5-20 mg / mL of an enzyme solution of glucose oxidase is sorted into the nanoelectrode to obtain a liquid-phase glucose oxidase-modified nanoelectrode.

7. The method of claim 1, wherein, the biomolecule to be detected in the cells is sphingomyelin oxidase, the reagent kit component is sphingomyelin, alkaline phosphatase, and choline oxidase, and a mixture containing 1-2 mM of sphingomyelin, 2-6 U / mL of alkaline phosphatase, and 2-6 U / mL of choline oxidase is sorted into the nanoelectrode to obtain a nanodroplet liquid-phase-modified nanoelectrode containing sphingomyelin oxidase reagent kit components.

8. Use of the method of any one of claims 1-7 in single-cell detection.

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