Preparation method and application of nano capillary tube with ion rectification effect

Abstract

The invention provides a preparation method of a nanocapillary with an ion rectification effect and application of the nanocapillary, and particularly relates to application of the nanocapillary in improving the resolution ratio, the image quality and the scanning speed of a scanning ion conductance microscope (SICM). Ions are rectified and modified on the nano capillary to improve the resolution of the SICM, and meanwhile, the image quality and the scanning speed are improved well.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of analytical testing and biomedicine, electrochemistry and materials science, and in particular relates to a nanocapillary with ion rectification effect, its preparation method and its application, especially the application of the nanocapillary in improving the resolution, image quality and scanning speed of scanning ion conductance microscopy (SICM). Background Technology

[0002] Scanning ion conductivity microscopy (SICM) is a non-contact, in-situ nanoscale surface morphology measurement instrument that has been widely used in biomedicine, electrochemistry, and materials science in recent years. SICM acquires images by utilizing the ionic current signal generated by the directional movement of ions in solution under the influence of an applied voltage, allowing it to obtain the surface morphology of a sample without physical contact. This makes it particularly suitable for non-destructive imaging of soft specimens, such as live cells. However, in practical applications, SICM exhibits some limitations. Redundant imaging time and reduced resolution: During SICM scanning, the probe moves forward and backward simultaneously. In traditional skip-mode scanning, the skip height is usually determined empirically. To avoid probe collisions with the sample, a high skip height is typically set during imaging, introducing unnecessary displacement, thus prolonging imaging time, reducing imaging efficiency, and limiting SICM's ability to perform in-situ dynamic observation of samples in practical applications. High scanning imaging noise: Within the same time interval, the faster the scanning speed, the faster the ion current changes. Excessive changes in ion current, exceeding the set attenuation threshold, may lead to the appearance of noise points. The faster the scanning speed, the greater the probability of noise appearing in the scanned image, which adversely affects image quality. Low imaging resolution: There is a trade-off between temporal resolution and image resolution. Therefore, pursuing higher temporal resolution at the same probe movement speed inevitably leads to a reduction in image resolution, limiting the SICM's ability to image detailed sample information.

[0003] As research into the microscopic structure and function of cells and biological structures in the biomedical field deepens, traditional imaging techniques such as optical and electron microscopy are gradually failing to meet researchers' resolution requirements. SICM, as an imaging technique providing nanoscale resolution, enables three-dimensional imaging of biological samples, thus possessing significant application value in cell biology, tissue engineering, and other fields. Improving imaging resolution has consistently been a research hotspot in SICM.

[0004] Existing methods have made considerable progress in some aspects. For example, to improve imaging resolution, measures such as adjusting the X and Y axes of the SICM, such as reducing the size of the nanocapillary tip or increasing the normal voltage applied to the probe, can enhance the probe tip and thus improve the lateral resolution of the SICM. By utilizing technologies such as piezoelectric ceramics, we can more precisely control the movement of the probe, thereby reducing the probe size while maintaining stable imaging quality.

[0005] However, the above methods also bring certain limitations and challenges: using smaller probe tips may require slower scanning speeds to obtain high-quality images, thus limiting imaging efficiency. Furthermore, sharper probes may damage soft or sensitive samples. Additionally, preparing extremely small probes may require complex processes and incur higher costs. Finally, maintaining consistency and reproducibility in tip size when preparing small probes in batches can be challenging. The same applies to increasing the ion current: increasing the probe voltage can increase the ion current in solution, resulting in smaller changes in the distance between the probe and the sample, producing more significant current changes, thereby improving imaging resolution. Simultaneously, it improves the system's sensitivity to distance changes, allowing the probe to respond more accurately to small fluctuations on the sample surface. Finally, within an appropriate voltage range, image quality can be improved, resulting in clearer images. However, increasing the voltage may increase the system's noise level, as higher voltages may lead to more electronic and background noise, thus damaging the nanocapillaries. Excessively high voltages may also damage the sample, especially when imaging soft or sensitive biological samples. Furthermore, increasing the voltage may affect the system's stability, particularly since maintaining a stable ion current at high voltages can be challenging. Finally, the increased voltage may exceed the design limits of the equipment and probes, leading to equipment performance degradation or damage, requiring equipment upgrades or replacements, thus increasing costs.

[0006] Given the limitations of existing methods, this invention aims to develop a novel approach to address the challenges of imaging resolution, specifically improving imaging efficiency, accuracy, and resolution while reducing cost, time requirements, and resource utilization. By adopting this strategy, we anticipate a significant improvement in the imaging resolution of SICMs, while simultaneously reducing costs and resource consumption during the imaging process. Therefore, this will provide a more efficient and precise imaging tool for fields such as biomedical research and materials science.

[0007] Ion rectification refers to the asymmetric transport characteristics of ions through certain materials or structures under an electric field, resulting in an asymmetric current-voltage (IV) characteristic, similar to the behavior of a diode in electronics. Its core principle lies in controlling ion flow using the asymmetric structure and surface properties of nanochannels. This phenomenon exhibits significant current-voltage characteristics under both forward and reverse voltages, similar to an electronic diode. Ion rectification is influenced by various factors, including geometrical channel asymmetry, surface charge distribution, biological effects, electrochemical regulation, and molecular screening mechanisms. Environmental conditions, such as pH, ionic strength, and temperature, also affect ion transport characteristics. Precise adjustment of these factors allows for precise control of ion flow, enabling the development of novel nanoscale devices in fields such as nanofluidics, biosensing, energy conversion, and storage. By applying this principle, under positive voltage conditions, a large amount of positive charge is distributed on the surface of a nanocapillary probe, enhancing the ion current while reducing redundant probe movement time during imaging. This method improves sample identification sensitivity by minimizing noise interference, ultimately enhancing SICM resolution along the Z-axis.

[0008] In summary, the method proposed in this invention, based on the principle of ion rectification and applied to nanocapillaries, improves the resolution of SICM imaging. Summary of the Invention

[0009] This invention provides a nanocapillary with ion rectification effect, its preparation method, and its applications, particularly the application of the nanocapillary in improving SICM resolution, image quality, and scanning speed. By modifying the nanocapillary with ion rectification, SICM resolution is improved, while image quality and scanning speed are also significantly enhanced.

[0010] Specifically, this method involves modifying a nanocapillary probe to induce an ion rectification effect. Compared to the unmodified probe, the modified probe carries a significantly positive charge on its surface. Under the influence of a positive voltage, it exhibits a higher current and a significantly improved rectification ratio than the unmodified probe. Furthermore, utilizing the modified probe helps to increase the Z-axis ion current and improve the Z-axis resolution.

[0011] This invention provides a nanocapillary with a rectifying effect, which is prepared by the following method:

[0012] a. First, use the P-2000 drawing instrument to draw nanocapillaries. The parameters are set as follows: Heat=310, Fil=3, Vel=10, Del=180, Pull=0. Line 2: Heat=320, Fil=3, Vel=20, Del=180, Pull=180.

[0013] b. After drawing, first clean it with a plasma cleaner, then activate it under the following conditions.

[0014] Prepare a mixed solution of NH3·H2O:H2O2:H2O = 1:1:5, then immerse the cleaned nanocapillaries in it and activate them for 30 minutes to expose the hydroxyl groups.

[0015] c. After activation, place it into the inner liner of a hydrothermal reactor, fix it to the edge of the inner liner, add 3-aminopropyltriethoxysilane diluted with anhydrous ethanol, seal the reactor, and react in an oven to obtain the final product.

[0016] Furthermore, in the nanocapillary preparation method of the present invention, the tip inner diameter of the nanocapillary pulled in step a is approximately 50 nm.

[0017] Furthermore, in the nanocapillary preparation method of the present invention, the soaking time in step b is 30 min.

[0018] Furthermore, in the nanocapillary preparation method of the present invention, the concentration of the solution in the reaction vessel in step c is 10% of the volume of APTES / anhydrous ethanol.

[0019] Furthermore, in the nanocapillary preparation method of the present invention, the reaction conditions in step c in the oven are: temperature 80°C and reaction time 1 hour.

[0020] Furthermore, the present invention provides a method for preparing nanocapillaries, characterized in that they are prepared by the following method:

[0021] a. First, use a P-2000 drawing instrument to draw nanocapillaries. The parameters are set as follows: Heat = 310, Fil = 3, Vel = 10, Del = 180, Pull = 0, line 2: Heat = 320, Fil = 3, Vel = 20, Del = 180, Pull = 180;

[0022] b. After the drawing is completed, it is first cleaned with a plasma cleaner, and then activated under the following conditions: prepare a mixed solution of NH3·H2O:H2O2:H2O=1:1:5, and then soak the cleaned nanocapillaries in it to activate them and expose the hydroxyl groups.

[0023] c. After activation, place it into the inner liner of a hydrothermal reactor, fix it to the edge of the inner liner, add 3-aminopropyltriethoxysilane, seal the reactor, and react in an oven to obtain the final product.

[0024] Furthermore, this invention also provides the application of nanocapillaries in improving the resolution, image quality, and scanning speed of scanning ion conductivity microscopy.

[0025] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0026] Compared to existing methods, the surface charge and material composition of the ion-rectifying nanocapillaries modified by this invention differ significantly from those of existing nanocapillaries, thereby improving the Z-axis resolution of scanning ion conductivity microscopy (SICM). This improvement reduces redundant probe movement, shortens imaging time, enhances sample identification sensitivity, and effectively reduces noise interference.

[0027] 1. Significantly improves Z-axis resolution and imaging sensitivity

[0028] By introducing a directional positive charge distribution through surface modification, the nanocapillaries exhibit a strong ion rectification effect under positive voltage. The modified probe generates a significantly enhanced ion current under the same positive voltage, greatly improving its sensitivity to micrometer / nanometer-scale undulations on the sample surface. This current enhancement directly translates into resolution optimization in the Z-axis direction, making it particularly suitable for high-precision three-dimensional morphology reconstruction of soft biological sample surfaces.

[0029] 2. Overcoming the limitations of the conflict between scanning speed and resolution

[0030] Traditional SICM requires reducing scanning speed or increasing jump height to avoid probe collisions, severely limiting imaging efficiency. The ion-rectifying nanocapillary of this invention, due to the directional guidance of ion flow by surface charge, can operate stably at a lower jump height, significantly shortening probe travel time. Experiments show that, while maintaining the same resolution, the scanning speed is increased, making in-situ observation of dynamic processes in living cells possible.

[0031] 3. Effectively suppresses imaging noise and improves the signal-to-noise ratio.

[0032] The charge-filtering effect created by the modified layer filters out background ion interference, while the rectification characteristics concentrate the current response within the positive voltage range. Compared to the unmodified probe, noise amplitude is reduced at the same scan rate, significantly improving the image signal-to-noise ratio. This characteristic is particularly suitable for high-resolution imaging in low-ionic-strength environments (such as cell culture media).

[0033] 4. Avoid high pressure damage and expand the compatibility of biological samples.

[0034] Existing technologies rely on increasing voltage to enhance resolution, but this can easily damage sensitive samples. This invention utilizes the surface charge-mediated ion enrichment effect to achieve equivalent current gain under conventional operating voltage, completely avoiding the risks of sample electrolytic damage and probe erosion caused by high voltage, and providing a safe window for long-term live-cell imaging.

[0035] 5. Reduce preparation costs and process complexity

[0036] Compared to traditional high-resolution probes that rely on physical diameter reduction at the tip (requiring precision drawing / etching), this approach achieves functional upgrades through surface chemical modification. The modification process is compatible with existing capillaries, requiring only one additional controllable modification step (such as polyelectrolyte layer-by-layer self-assembly) to transform conventional nanocapillaries into high-performance ion rectifying probes, significantly reducing fabrication costs and ensuring batch consistency.

[0037] 1) The method proposed in this invention significantly advances the development of SICM in high-resolution cell imaging by addressing issues related to poor image quality and simultaneously improving scanning speed without compromising image quality. Notably, the increased scanning speed provides potential value for in-situ dynamic sample observation.

[0038] 2) This invention not only improves the efficiency, accuracy and resolution of SICM imaging, but also reduces cost, time and resource consumption. Attached Figure Description

[0039] Figure 1 (a) SEM image of the nanopipette. (b) IV curves as the nanopipette structure changes, with the inset showing the corresponding changes in the ICR ratio.

[0040] Figure 2 (a) Energy spectrum of unmodified nanocapillaries; (b) Energy spectrum of modified nanocapillaries.

[0041] Figure 3 (a) Rectification ratio of nanocapillaries at different temperatures 。 (b) Comparison of cyclic voltammetry (CV) curves of APTES-modified nanocapillary surfaces under different silanization conditions. Data for each group are the average of three independent samples (n=3), with standard deviations (SD) indicated.

[0042] Figure 4 (a) Imaging of modified nanotubes on a glass substrate, (b) Imaging of unmodified nanotubes. (c) Standard deviation of height (SD) of different nanofluidic capillaries scanning a glass substrate under the same conditions (n ​​= 1024), with SD of 0.01553 for unmodified nanotubes and 0.002913 for APTESS.

[0043] Figure 5 In the figure, a, b, and c represent the cell imaging effects of unmodified nanocapillaries at different thresholds (0.015, 0.02, and 0.03), respectively; d, e, and f correspond to the imaging results of modified nanocapillaries under the same threshold conditions. Among the comparison groups a and d, b and e, and c and f, all experimental conditions were kept consistent except for the threshold parameters in the SICM settings.

[0044] Figure 6 In the diagram, a, b, c, and d are schematic diagrams of cell imaging using modified nanofluidic tubes, while e, f, g, and h are schematic diagrams of imaging using the same device. Specifically, a and e use a scan rate of 6 V / s; b and f use a scan rate of 10 V / s; c and g use a scan rate of 15 V / s; and d and h use a scan rate of 20 V / s.

[0045] Figure 7 a represents the current trajectory of a cell scanned using an unmodified nanofluidic capillary tip; b represents the current trajectory of a cell scanned using a modified nanofluidic capillary tip.

[0046] Figure 8 The surface modification process of the nanocapillary is clearly shown from left to right: ① Original state: The surface of the unmodified glass tube is electrically neutral (no net charge), and the ion current is linear (no rectification); ② Activated state: The exposed hydroxyl groups (-OH) on the surface are negatively charged, inducing negative rectification (I0). 负 >I 正 ); ③ Functional state: APTES-modified amino groups (-NH2) form a positively charged layer on the surface, achieving strong positive rectification (I 负 正 ).

[0047] Abbreviations:

[0048] Scanning Ion Conductance Microscopy (SICM)

[0049] 3-Aminopropyltriethoxysilane: APTES

[0050] Cyclic voltammetry (CV)

[0051] Ion current rectification (ICR)

[0052] Scanning electron microscope (SEM)

[0053] Human normal mammary epithelial cells, MCF-10A

[0054] Phosphate-buffered saline (PBS) Detailed Implementation ​

[0055] The following experiments and examples are used to further illustrate, but are not limited to, the present invention.

[0056] Experimental Section

[0057] Reagents and materials

[0058] Fetal bovine serum was obtained from SAOMAI. 1640 medium, PBS cell buffer, and trypsin digestion enzymes were all from Solarbio. APTES were provided by McLean. KCL powder was provided by Sangon Biotech. The incubator was manufactured by Chuanhong Shanghai Co., Ltd. The hydrothermal reaction vessel originated from Shangyi City. The P-2000 traction device and nanotube (BF100-58-10) were products of Sutter Instruments (CA). MCF-10A cells were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Throughout the experiment, ultrapure water with a resistivity of 18.2 MΩ cm was used, and buffer solutions were sterilized.

[0059] Cell culture: MCF-10A (normal human mammary epithelial cells) were cultured in 1640 cells supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cells were cultured at 37°C in a 5% / 95% CO2 / air incubator.

[0060] Fabrication of nanocapillaries: Nanocapillaries with diameters of 50 nm to 100 nm were fabricated using a P-2000 microcapsule puller (Sutter instrument). The morphology of the nanocapillaries was characterized using scanning electron microscopy (SEM, Hitachi S-4800, Japan).

[0061] The borosilicate nanotubes used in this embodiment were (BF100-58-10, Sutter Instruments, USA).

[0062] The P-2000 program contains the following parameters: Heat=310, Fil=3, Vel=10, Del=180, Pull=0; line 2: Heat=320, Fil=3, Vel=20, Del=180, Pull=180

[0063] Characterization of nanoparticles: The fluctuation of the nanotube pulse time should be controlled within 6 s to ensure the uniformity of the nanotube geometry. For easier observation, gold (HIYACHI MC1000) must be coated on the nanotubes to improve their conductivity. The nanotubes were characterized by scanning electron microscopy (SEM) on a SU8020 instrument (Hitachi).

[0064] Example 1: Modification of nanocapillaries and exploration of optimal modification conditions

[0065] After hydroxylation and washing with deionized water, the concentration of APTES (5%) and modification time (60 min) remained initially constant. The optimal temperature for the nanocapillary was explored by comparing the CV values ​​and ion current rectification (ICR) at 50 °C, 60 °C, 70 °C, and 80 °C. Finally, the optimal modification times (15 min, 30 min, 60 min, 120 min) and concentrations (1%, 5%, 10%, 20%) were determined based on the measured CV values ​​and ICR. Excess solution was then thoroughly rinsed with ethanol and deionized water.

[0066] Table 1. Rectification ratio measurements of APTES-modified nanoparticles at different reaction times under different silanization conditions. For each case, the reported values ​​are the average rectification ratios from the same batch of nanoparticles, along with the standard deviation (SD).

[0067]

[0068] Table 1 shows that the ion current rectification ratio (ICR) of the nanocapillary changes nonlinearly with the APTES concentration (1%-20%) and reaction time (15-120 minutes). The larger the ICR value, the stronger the ion rectification effect—that is, the probe's ability to conduct ion current in the forward direction far exceeds its ability to limit current in the reverse direction (similar to an electronic diode).

[0069] The core principle is:

[0070] ① Optimal process combination: When reacting at a concentration of 10% APTES for 60 minutes, the ICR reached 15.43±0.61 (15 times that of the unmodified probe), proving that this parameter can form a dense monomolecular charge layer;

[0071] ② Concentration threshold window: ICR>5 can only be achieved in the 8-12% concentration range. Rectification is weak (ICR≤2.38) below 5%, while ICR decreases by 29% compared to the 10% group due to channel blockage caused by molecular stacking at 20% concentration.

[0072] ③ Time-sensitive peak: 60 minutes is the performance inflection point. If it is too short (15 minutes), the modification is insufficient (ICR≤1.31). If it is too long (120 minutes), the ICR will decrease by 67% due to silane hydrolysis.

[0073] ④ Process stability: The 10% concentration group had the lowest standard deviation (±0.61) and a coefficient of variation of only 3.9%, significantly better than the 20% group (±0.78). This data reveals that the ultra-strong rectification effect of ICR=15.43 is the technical cornerstone of this invention, which directly promotes the improvement of the Z-axis resolution of SICM by amplifying the forward current and suppressing reverse interference.

[0074] Surface modification of nanotubes: APTES modification was performed to achieve uniform coverage of positive charge on the tip surface. Prior to this modification, organic impurities were removed from the surface. Subsequently, to increase the exposure of hydroxyl groups and improve modification efficiency, the samples were immersed in a mixed solution of ammonia, hydrogen peroxide, and water (volume ratio 1:1:5) and incubated with shaking at room temperature for 30 minutes. After incubation, they were washed three times with deionized water and then dried in an oven. The nanotubes were then placed in a hydrothermal reactor containing a 10% v / v APTES ethanol solution and reacted at 80°C for 1 hour. The principle and final rectification effect are shown in the figure below. Figure 8 As shown.

[0075] SICM Setup: SICM imaging was performed using a scanning module integrated inverted fluorescence microscope (Leica, Germany & Crystal Microscope, China). The system consisted of an XY plane scanner (40 μm x 40 μm) and a probe equipped with a 40 μm Z-axis scanner. A glass microtube was fixed to the head as the SICM probe; this microtube was made of borosilicate capillary material, with an inner diameter of approximately 50 nm, and was pulled out using a CO2 laser-based microtube puller. The pipette was filled with phosphate-buffered saline (PBS) and an Ag / AgCl electrode was inserted. For SICM operation, the sample on a coverslip was placed in the PBS-filled cell chamber, and imaging was performed in jump mode. In this mode, ion currents were recorded as the pipette was moved vertically to repeatedly approach and retract from the sample surface. The threshold for stopping the pipette method was set to increase the resistance by 2% relative to the baseline resistance. The SICM setup facilitated the measurement of ion currents, providing feedback to control the distance between the nanopipette tip and the sample surface. A complete image of the sample surface was obtained by scanning with the nanotube.

[0076] Example 2: Image Quality Comparison Method

[0077] To achieve more precise quantification of imaging quality, modified and unmodified nanotubes will be used to scan an empty culture dish with a flat glass bottom, allowing for comparison of the height standard deviation between the two surfaces. Furthermore, MCF-10A cells will be scanned simultaneously under consistent conditions, facilitating comparisons of cell imaging at different threshold levels (0.015, 0.02, 0.03). Finally, to illustrate the improved imaging quality, whole-cell and localized region scans will be performed to compare the imaging quality of the two methods.

[0078] Imaging speed comparison: In addition, to accurately quantify the imaging speed of SICM, cell samples were imaged at different scan speeds (6, 10, 16 and 20 V / s) while keeping all parameters consistent; the resulting images were then compared.

[0079] Example 3: Scanning electron microscopy analysis of nanocapillaries

[0080] Scanning electron microscopy (SEM) revealed that the pristine nanotube exhibited a nearly cylindrical nanotip. Figure 1 ) and a nearly circular aperture of approximately 50 nm ( Figure 1 a). Based on its inherent ICR properties, the stepwise modification of nanotubes was carefully monitored. As shown in the figure, the original nanotubes produced a minimal ICR effect ( Figure 1 b, black curve), while the surface of the plasma gasket is subject to significant negative charge accumulation (yellow curve);

[0081] APTES functionalization dramatically altered the surface charge, transforming it from a positively charged suspended amine to a more neutral state (red curve). As expected, due to the presence of positively charged amine groups within the APTES coverage area, the surface of the nanocapillaries exhibited a high density of positively charged species (red curve). The corresponding ICR ratios were then calculated ( Figure 1 (b illustration), here defined as the absolute value of I+0.5V / I-0.5V.

[0082] Example 4: Nanotube Voltammetry Analysis and Optimization of Modification Conditions

[0083] First, we maintained an APTES concentration of 5% and a reaction time of 60 minutes. Then, we compared the CV values ​​at different reaction temperatures (50℃, 60℃, 70℃, and 80℃) to assess the rectification effect and explore the optimal modification temperature. We measured the rectification value of the nanocapillaries using cyclic voltammetry (CV) mode in SICM. This measurement depends on the density of positive and negative charges on the nanocapillary surface. We measured and plotted the CV changes at different concentrations to demonstrate the relationship between CV change and APTES concentration. During the reaction at 80℃, we selected six concentrations of APTES (1%, 5%, 10%, and 20%). The results showed that the CV change was greatest when the APTES concentration was 10%.

[0084] Example 5: Comparison of Imaging Results

[0085] First, we performed scanning tests on glass culture dishes with smooth substrates. For example... Figure 4 As shown in a and b, the modified nanofluidic capillary (standard deviation 0.4451 ± 0.002913) has a significantly lower standard deviation than the unmodified one (0.4349 ± 0.01553). Furthermore, while maintaining consistent SICM parameters, the modified nanofluidic capillary exhibits lower noise and higher imaging stability.

[0086] Subsequently, we used SICM technology to image MCF-10A cells in confocal culture dishes. While keeping other SICM parameters constant, we adjusted the thresholds (0.015, 0.02, 0.03). From Figure 5 It can be seen that compared to the performance of d and a at a threshold of 0.015, the unmodified probe, due to its lower current and closer proximity to the sample, generates significantly more noise during imaging, ultimately affecting the imaging effect. While the imaging quality of the unmodified probe improves with increasing threshold, noise issues still exist, and the imaging effect of b is significantly inferior to e. Therefore, modification and rectification significantly enhance the imaging resolution of SICM nanocapillaries in the Z-axis direction. We used SICM to image MCF-10A cells on confocal culture dishes. While keeping other SICM conditions constant, we varied the threshold (0.015, 0.02, 0.03). Figure 5 It can be inferred that at a threshold of 0.015 ( Figure 5 a and 5d), unmodified probe ( Figure 5 a) Due to the weak ion rectification effect resulting in a smaller current, and the probe's tendency to over-approach the sample in hopping mode, the generated noise is significantly higher than that of modified probes with a rectification effect. Figure 5 (d) The imaging effect is significantly affected by the increase of the threshold setting. Although the imaging of unmodified nanocapillaries is improved to some extent, noise still exists, and the imaging effect of b is significantly lower than that of e. Therefore, it can be observed that after modification and obtaining the correction effect, the imaging resolution of nanocapillaries on the Z-axis of SICM is significantly improved.

[0087] Example 6: Comparison Results of Imaging Speed

[0088] MCF-10A cells were imaged on confocal culture dishes using SICM. While keeping other SICM conditions constant, we varied the scan rate (6V / s, 10V / s, 15V / s, 20V / s). From Figure 6 It can be inferred that the scanning speeds of a and e are both 6V / s, and their imaging effects are similar. With increasing threshold settings, at 15V / s, the imaging of unmodified nanocapillaries (g) shows a certain degree of whealing, and the imaging effect is significantly lower than that of c. In addition, Figure 7 The current and time data also show that while increasing the current, the imaging time is significantly shortened from 5 minutes to 1 minute. This demonstrates that after modification and rectification, the imaging speed of the nanofluidic tube on the Z-axis of the SICM is significantly improved.

[0089] The above modifications, through surface charge reconstruction, can be prepared in a short time and made affordable to achieve ultra-high resolution within 100 nanometers, while breaking through the time and cost constraints of the traditional "diameter reduction and material replacement" approach.

[0090] This study effectively improved the Z-axis resolution of scanning ion conductivity microscopy (SICM) by applying the ion rectification effect. Modification of the surface of borosilicate-silica nanocapillaries with APTES generated a large amount of positive charge, significantly enhancing the ion rectification effect. In terms of imaging quality and speed, the improved nanocapillaries showed significant improvements in both ion current and imaging efficiency due to the increased ion current from ion rectification. This enhancement reduced redundant probe movement, shortened imaging time, improved sample identification sensitivity, and minimized noise interference. Therefore, the method employed in this study not only improved imaging efficiency, accuracy, and resolution but also reduced cost, time, and resource consumption. It shows considerable promise in advancing high-resolution cell imaging using SICM and provides valuable potential for in-situ dynamic observation of samples.

[0091] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A nanocapillary having a rectifying effect, characterized by, The following preparation method is used to prepare: a. First, use P-2000 drawing instrument to draw nanocapillary, and the parameters are set as follows: Heat=310, Fil=3, Vel=10, Del=180, Pull=0, line 2: Heat=320, Fil=3, Vel=20, Del=180, Pull=180; b. After drawing, first use plasma cleaning machine to clean it, then activate it by using the following conditions: configure a mixed solution of NH3·H2O:H2O2:H2O=1:1:5, and then immerse the cleaned nanocapillary in it to activate it and expose the hydroxyl groups; c. After activation, put it into the inner container of the hydrothermal reactor, fix it on the edge of the inner container, add 3-aminopropyl triethoxysilane diluted with anhydrous ethanol, seal the reactor, and react in the oven to obtain it.

2. The nanocapillary of claim 1, wherein, The inner diameter of the needle tip of the nanocapillary drawn in step a is about 50 nm.

3. The nanocapillary of claim 1, wherein, The immersion time in step b is 10-60 min.

4. The nanocapillary of claim 3, wherein, The immersion time in step b is 30 min.

5. The nanocapillary of claim 1, wherein, The concentration of the solution in the reactor in step c is 10% of the volume of APTES / anhydrous ethanol.

6. The nanocapillary of claim 1, wherein, The reaction conditions in the oven in step c are: temperature 80°C, reaction time 1 hour.

7. The method for preparing nanocapillaries according to claim 1, characterized in that, The following preparation method is used to prepare: a. First, use P-2000 drawing instrument to draw nanocapillary, and the parameters are set as follows: Heat=310, Fil=3, Vel=10, Del=180, Pull=0, line 2: Heat=320, Fil=3, Vel=20, Del=180, Pull=180; b. After drawing, first use plasma cleaning machine to clean it, then activate it by using the following conditions: configure a mixed solution of NH3·H2O:H2O2:H2O=1:1:5, and then immerse the cleaned nanocapillary in it to activate it and expose the hydroxyl groups; c. After activation, put it into the inner container of the hydrothermal reactor, fix it on the edge of the inner container, add 3-aminopropyl triethoxysilane, seal the reactor, and react in the oven to obtain it.

8. The application of the nanocapillary of claim 1 in improving the resolution, image quality and scanning speed of scanning ion conductance microscopy.

9. The application of claim 1, wherein the nanocapillary with rectification effect is obtained by modifying borosilicate nanocapillary by ion-electron rectification method.

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