PH probe based on living cell incubation as well as preparation method and application of pH probe

Abstract

The invention discloses a pHi probe based on living cell incubation as well as a preparation method and application of the pHi probe. The preparation method comprises the following steps: drawing a capillary tube into a nano suction tube with the inner diameter of 120 nm; inserting the prepared nano-pipette into cells, incubating for a period of time, then washing the nano-pipette with ultrapure water, then putting the nano-pipette into a phosphate buffer solution (PBS) with phosphate radicals removed, and preserving, so as to obtain the pHi probe based on living cell incubation after the preservation is finished. According to the preparation method of the pHi probe disclosed by the invention, the pHi probe is prepared by inserting the nano suction tube into the cell, and the pHi probe can be prepared by inserting the nano suction tube into the cell and incubating for 60 minutes. The preparation method is simple, efficient and low in dependence on equipment, the prepared nanopipette pHi probe is sensitive to pH response, and the dynamic change of single cell pHi can be monitored in real time.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a pHi probe based on live cell incubation, its preparation method, and its application. Background Technology

[0002] As the basic unit of life, the cell's intracellular pH (pHi) is crucial for maintaining normal physiological functions and metabolism. It directly affects multiple key cellular processes, including protein function, cell metabolism, growth, proliferation, and migration. Accurate detection of pHi is of great significance for a deeper understanding of cellular pathophysiological mechanisms, early disease diagnosis, and efficient drug screening and pharmacokinetic studies.

[0003] In recent years, the development of single-cell pHi probe technology has provided an important tool for cell research. Electrochemical probe technology based on nanopores / nanopipettes has shown significant advantages: its nanoscale tips can penetrate cells with low invasiveness and achieve high spatiotemporal resolution pHi sensing in single cells. Its sensing basis stems from the response of the ion transport behavior of the nanopore tip to external stimuli (such as pH). To enhance pH response, researchers commonly employ strategies of modifying the inner wall with chemical groups and filling with pH-sensitive materials, achieving significantly enhanced pH sensing capabilities. However, current nanopipette pHi probes based on inner wall modification and pH-sensitive material filling still face significant challenges, such as the difficulty, low success rate, and poor sensitivity of the modification process, which seriously affect the reliability of intracellular pH detection. Furthermore, the need for exogenous molecular modification and the complexity and low efficiency of preparation methods further limit their practical application in real-time monitoring of live single cells. Summary of the Invention

[0004] This invention provides a pHi probe based on live cell incubation, its preparation method, and its application, in order to overcome the above-mentioned problems.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a method for preparing a pHi probe based on live cell incubation, comprising the following steps: S1: The capillary tube is drawn into a nanotube with an inner diameter of 120 nm; S2: The nanopipette prepared in S1 was inserted into the cell and incubated for a period of time. Then, the nanopipette was rinsed with ultrapure water and then placed in a phosphate-free PBS solution for preservation. After preservation, the pHi probe based on live cell incubation was obtained.

[0006] Furthermore, in S2, the incubation time is 10 min to 90 min.

[0007] Furthermore, in S2, the cell types include human glioblastoma cells T98G, human renal epithelial cells 293T, human liver cancer cells HCCLM3, human prostate cancer cells DU-145, microglia BV2, and placental villous trophoblast cells HTR-8.

[0008] Furthermore, in S1, the dimensions and material of the capillary are as follows: Borosilicate capillary tube BF100-59-10 with an inner diameter of 0.59 mm and an outer diameter of 1.00 mm; Borosilicate capillary tube BF120-69-15 with an inner diameter of 0.69 mm and an outer diameter of 1.20 mm; Borosilicate capillary tube BF100-78-15 with an inner diameter of 0.78 mm and an outer diameter of 1.00 mm; Borosilicate capillary tube BF150-86-10 with an inner diameter of 0.86 mm and an outer diameter of 1.50 mm; Quartz capillary tube BF100-70-7.5 with an inner diameter of 0.70 mm and an outer diameter of 1.00 mm.

[0009] Furthermore, in S1, the parameters for drawing the capillary are as follows: Borosilicate capillary tube BF100-59-10: Heat=280, Fil=0, Vel=20, Del=145, Pull=150; Borosilicate capillary tube BF120-69-15: Heat=350, Fil=0, Vel=10, Del=145, Pull=160; Borosilicate capillary tube BF100-78-15: Heat=400, Fil=0, Vel=20, Del=145, Pull=180; Borosilicate capillary tube BF150-86-10: Heat=350, Fil=0, Vel=20, Del=145, Pull=160; Quartz capillary tube QF100-70-7.5: Heat=650, Fil=0, Vel=20, Del=145, Pull=220.

[0010] Furthermore, the phosphate-removing PBS solution comprises 137 mM NaCl and 2.7 mM KCl, with a pH of 6.82.

[0011] In another aspect, the present invention provides a pHi probe based on live cell incubation, wherein the pHi probe based on live cell incubation is prepared by the aforementioned preparation method.

[0012] The final aspect of this invention provides the application of the described pHi probe based on live cell incubation in detecting intracellular pH.

[0013] The beneficial effects of this invention are: This invention discloses a method for preparing a pHi probe based on live cell incubation. The pHi probe is prepared by inserting a nanoparticle into a single cell for incubation. The method is simple, efficient, does not require additional biochemical reagents for functionalization, and the prepared pHi probe has high sensitivity and can monitor the dynamic changes of pHi in single cells in real time. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is an electron microscope image of the nanostraw prepared in Example 1 of the present invention; Figure 2 This is a graph showing the pH response behavior of the pHi probe prepared in Example 1 of the present invention based on ion current rectification. Figure 3 The pH response curve of the pHi probe prepared in Example 1 of this invention based on the ion current rectification ratio (n=6). Figure 4 The image shows the IV curves (n=6) of the reversible response of the pHi probe prepared in Example 1 of this invention to two different pH solutions, pH 2.93 and pH 9.11. Figure 5 The rectified ratio oscillation diagram (n=6) shows the reversible response of the pHi probe prepared in Example 1 of this invention to two different pH solutions, pH2.93 and pH9.11. Figure 6 This is a statistical graph showing the rectification ratio of the pHi probe prepared in Example 1 of the present invention against common intracellular interfering substances. Figure 7 The graph shows the effect of cell incubation time on the pH response sensitivity of the pHi probe. Figure 8 pH response sensitivity of pHi probes prepared by incubation with different cell types based on rectification ratio; Figure 9 A comparison of the pH response sensitivity of pH probes prepared by incubating capillaries of different materials and models in DU-145 cells for 60 min. Figure 10 This is a micrograph of the pHi probe prepared in Example 1 of the present invention inserted into DU145 cells for pHi measurement; Figure 11 The graph shows the current change when the pHi probe prepared in Example 1 of this invention is inserted into DU145 cells for pHi measurement. Figure 12 This is a comparison diagram of the pH values ​​inside and outside DU145 cells measured by the pHi probe prepared in Example 1 of this invention; Figure 13 The rectified ion current curves of the pHi change in DU145 cells before and after the addition of ammonium chloride solution, as detected by the pHi probe prepared in Example 1 of this invention; Figure 14 This is a statistical graph (n=6) showing the rectifier ratio of pHi changes in DU145 cells before and after the addition of ammonium chloride solution, as detected by the pHi probe prepared in Example 1 of this invention. Figure 15 The ion current rectification curves of the pHi change in DU145 cells before and after the addition of sodium acetate solution, as detected by the pHi probe prepared in Example 1 of this invention; Figure 16 This is a statistical graph (n=6) showing the rectifier ratio of the pHi change in DU145 cells before and after the addition of sodium acetate solution, as detected by the pHi probe prepared in Example 1 of this invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example: Borosilicate capillary tubes (BF 100-59-10) with an outer diameter of 1.00 mm and an inner diameter of 0.59 mm were drawn into nanotubes with an inner diameter of 120 nm using a P-2000 microelectrode laser drawing instrument. The drawing parameters were Heat=280, Fil=0, Vel=20, Del=145, and Pull=150.

[0017] The nanopipette, drawn to an inner diameter of 120 nm, was inserted into DU-145 cells using a micromanipulation system (MP-365) and incubated for 60 minutes. After thoroughly rinsing the nanopipette with ultrapure water, it was stored in a centrifuge tube containing phosphate-free PBS solution (137 mM NaCl, 2.7 mM KCl, pH 6.82) to obtain the pHi probe based on live cell incubation.

[0018] Performance testing of the pHi probe: (1) pH response experiment: method: A. The pH response behavior of the nanopipettes prepared in the examples was tested using an electrochemical workstation (CHI830D). Phosphate-free PBS solution was injected into the tail end of the nanopipette using a microsyringe to remove air bubbles. B. A 0.3 mm diameter Ag / AgCl reference electrode was inserted into the end of a nanocapillary as the working electrode, and another Ag / AgCl reference electrode was inserted into the electrolyte buffer as both the counter and reference electrodes. Linear sweep voltammetry (-1.0 V to +1.0 V, 0.1 V / s) was used to record the ion current, and data was recorded after stabilization. The rectification ratio r (r = log2|I) + / I - | ), used to quantify the direction and intensity of ion current rectification (ICR). The pH response capability of the probe was validated using a series of phosphate-buffered saline (PBS) solutions with pH values ​​of 2.93, 3.98, 5.01, 5.89, 6.87, 8.01, and 9.11. The PBS contained 137 mM NaCl, 2.7 mM KCl, and 10 mM PO4. 3+ The solution; DU-145 cells in the logarithmic growth phase were discarded from their culture medium, rinsed three times with 1×PBS, and then 1×PBS was added again. Cell pHi was measured using a microscope, a micromanipulation system (MP-365), and an electrochemical workstation (CHI830D). The entire setup was placed on an optical vibration-damping platform to minimize interference from environmental vibrations. After inserting the prepared pHi probe into adherent DU-145 cells in a culture dish, one Ag / AgCl electrode (0.3 mm) was placed in the culture solution as both the counter and reference electrode. Another Ag / AgCl electrode was inserted into the lumen of a nanopipette from its rear end as the working electrode. The current changes during cell puncture were recorded in iterative mode, and the pH difference between the inside and outside of the cells was recorded in inductively coupled plasma (IVP) mode.

[0019] result: Electron microscopy was performed on the nanoparticles prepared in the examples, and the results are shown in the figure. Figure 1As can be seen from the figure, the inner diameter of the nanostraw drawn in this embodiment is 120 nm; Figure 2 The following is an IV curve of the pHi probe prepared in the example at different pH solutions. Figure 2 As can be seen, the pHi probe exhibits IV curves with different ICR directions and intensities in solutions of different pH values, indicating that the pHi probe has pH response capability based on ICR. Figure 3 The graph shows the linear relationship between the rectification ratio and solution pH of the ICR response of the pHi probe prepared in the examples in solutions with different pH values. Figure 3 As can be seen, the rectification ratio of the pHi probe decreases with increasing solution pH. Linear fitting shows a good linear relationship between the rectification ratio of the pHi probe and the solution pH within the range of pH 2.93 to pH 9.11 (r = -1.03108pH + 5.80067; Ri). 2 = 0.99705; n = 6); Figure 4 The image shows the IV curves (n=6) of the reversible response of the pHi probe to two different pH solutions, pH 2.93 and pH 9.11. Figure 4 As can be seen, the pHi probe has different ICR directions in two different pH solutions with pH 2.93 and pH 9.11. When the solution pH is changed repeatedly, the ICR direction changes repeatedly. Moreover, the ICR overlaps well in the same pH solution, indicating that the pHi probe has good pH response reversibility. Figure 5 The plot shows the oscillation of the rectification ratio (n=6) of the reversible response of the pHi probe to solutions with different pH values ​​of pH 2.93 and pH 9.11. Figure 5 As can be seen, the pHi probe has different rectification ratios in two pH solutions: pH 2.93 and pH 9.11. When the pH value of the solution is changed repeatedly, the rectification ratio fluctuates repeatedly, indicating that the pHi probe has good pH response reversibility. (2) Sensitivity experiment of pHi probe to interfering substances: Multiple interfering substances, at a pH of 6.87, were continuously layered at 50 μM. These substances included salts of common anions and cations (KCl, NaCl, MgCl2, CuCl2, FeCl3, AlCl3, NH4Cl, CaCl2, NaHCO3) and small biological molecules (ATP, GSH, Glutamic acid, Ascorbic acid, L-cysteine, and Glucose). + A concentration of PBS buffer (pH 4.3) was used as a control. Results are as follows: Figure 6 The results show that the pHi probe is insensitive to the rectification ratio in common interfering substances, but exhibits a significant change in the ion current rectification ratio in hydrogen ion solutions of the same concentration, indicating that the pHi probe has good pH response selectivity.

[0020] (3) Experiment on the effect of different incubation times on the pH response sensitivity of the prepared pHi probe based on the rectification ratio: Nanoparticles with an inner diameter of 120 nm, drawn from BF 100-59-10 capillary tubes, were inserted into DU145 cells and incubated for 10 to 90 minutes (10 min, 15 min, 20 min, 30 min, 50 min, 60 min, and 90 min) to prepare pHi probes (cell group). pH response sensitivity was tested, and nanoparticles with an inner diameter of 120 nm, drawn from BF 100-59-10 capillary tubes (bare tube group), were used as a control. Results are as follows: Figure 7 As shown in the figure, the pH response sensitivity of the pHi probe gradually increases with the extension of incubation time, reaching a maximum value (1.01 pH) at 60 min. -1 When the time was extended to 90 minutes, no significant improvement in sensitivity was observed.

[0021] (4) Experiment to investigate the effect of different cell types of incubation on the pH response sensitivity of the prepared pHi probe based on the rectification ratio: Nanoparticles with an inner diameter of 120 nm, drawn from BF 100-59-10 capillary tubes, were inserted into human glioblastoma cells T98G, human renal epithelial cells 293T, human hepatocellular carcinoma cells HCCLM3, human prostate cancer cells DU-145, microglia BV2, and placental villous trophoblast cells HTR-8 for 60 min to prepare nanoparticle-based pHi probes. Their pH response sensitivity based on the rectification ratio was measured, and the results are as follows: Figure 8 As shown, the nanopipettes incubated with six different cell lines all exhibited highly consistent pH response sensitivity, indicating that the pHi probe prepared by this method can effectively construct sensitive pH-responsive interfaces in different cell lines.

[0022] (5) Experiment on the effect of different materials and models of capillary tubes on the pH response sensitivity of pH probes prepared by incubation in DU-145 cells for 60 min: Capillaries were drawn according to the drawing parameters shown in Table 1 below, and probes were prepared according to the methods described in the above embodiments. The effect of pH response sensitivity was tested, and the results are as follows: Figure 9As shown, all five types of nanopipettes produced pHi probes exhibited significant and highly consistent improvements in pH response sensitivity, with sensitivity values ​​of 1.01±0.08, 1.07±0.13, 0.98±0.11, 0.98±0.08, and 1.02±0.08 pH, respectively. -1 The results show that, regardless of the original capillary material (borosilicate or quartz) or specific model, as long as it is successfully drawn into a nanopipette, the enhanced pH response induced by cell incubation can be stably reproduced, fully verifying the good universality of this method across capillary types.

[0023] Table 1. Capillary tubes of different materials and models and their drawing parameters

[0024] (6) The pHi and pHe values ​​of DU145 cells were measured using a pHi probe (model BF 100-59-10 borosilicate capillary, prepared by pulling down parameters Heat=280, Fil=0, Vel=20, Del=145, Pull=150, and then incubated in DU-145 cells for 60 minutes). The prepared pHi probe was inserted into DU145 cells and pHi was measured. Microscopic images are shown below. Figure 10 As shown, when the pHi probe was inserted into DU-145 cells, the cell morphology did not change significantly, indicating that the pHi probe prepared by this method is a nanoprobe with low cell damage.

[0025] Figure 11 This graph shows the current changes measured by pHi when the pHi probe is inserted into DU145 cells. Figure 11 The results showed that the stepwise changes in current before and after the pHi probe was inserted into DU-145 cells demonstrated the precise insertion and extraction of the cell probe.

[0026] Figure 12 A comparison of intracellular and extracellular pH values ​​measured by the pHi probe in DU145 cells, from... Figure 12 As can be seen, the extracellular pH (pHe) value measured by the pHi probe before penetrating DU-145 cells was 6.9±0.22, and the intracellular pH (pHi) value measured by the pHi probe after penetrating the cells was 7.45±0.25.

[0027] (7) The pHi probe (model BF 100-59-10 borosilicate capillary was prepared by pulling down the parameters Heat=280, Fil=0, Vel=20, Del=145, Pull=150 and then incubating in DU-145 cells for 60 minutes to obtain the pHi probe) was subjected to a drug-induced intracellular pH dynamic change experiment: DU-145 cells in the logarithmic growth phase were discarded from their culture medium, rinsed three times with 1×PBS, and then 1×PBS was added. Cell pHi was measured using a microscope, a micromanipulation system (MP-365), and an electrochemical workstation (CHI830D) (the entire setup was placed on an optical plate with an airbag underneath for shock absorption). After inserting the prepared pHi probe into the cells, 20 mol / L ammonium chloride was slowly added to the culture dish containing DU-145 cells until a final concentration of 10 mol / L was reached, followed by incubation. Simultaneously, 20 mol / L sodium acetate solution was slowly added until a final concentration of 10 mol / L was reached as a control group to adjust cell pHi. One Ag / AgCl electrode (0.3 mm) was placed in the culture dish solution as both the counter and reference electrode. Another Ag / AgCl electrode was inserted into the lumen of a nanopipette from the rear end as the working electrode. The pHi changes of DU-145 cells before and after the addition of the drugs (ammonium chloride and sodium acetate) were recorded using IV mode.

[0028] Figure 13 The ion current rectification curves for pHi changes in DU145 cells before the addition of ammonium chloride solution (before incubation in the figure) and after the addition of ammonium chloride solution (after 30 min of ammonium chloride incubation in the figure) were obtained by detecting the pHi probe. Figure 13 As can be seen, the addition of ammonium chloride solution to the PBS solution used to culture DU-145 cells increased the ICR of the pHi probe, indicating that the pHi of DU-145 cells increased.

[0029] The rectification ratio of pHi changes in DU145 cells before and after the addition of ammonium chloride solution was detected using a pHi probe. Statistical graphs (n=6) are shown below. Figure 14 As shown, from Figure 14 As can be seen from the data, the rectification ratio of six independent DU-145 cells increased after the addition of ammonium chloride solution, indicating that the cell pHi increased in the presence of ammonium chloride. The cell pHi increased from 7.42±0.17 to 7.78±0.10, which shows that the pHi probe prepared in this invention can monitor drug-induced pHi changes.

[0030] Figure 15The figure shows the ion current rectification curves for pHi changes in DU145 cells before and after the addition of sodium acetate solution, as detected by the pHi probe. As can be seen from the figure, the ICR intensity of the pHi probe decreased after adding sodium acetate solution to the PBS solution used to culture DU-145 cells. This result indicates that the pHi of DU-145 cells decreased. This demonstrates that the pHi probe prepared by cell incubation can be used to monitor dynamic changes in intracellular pH. (The figure shows the ion current rectification curves for pHi changes in DU145 cells before adding sodium acetate solution, and the figure shows the ion current rectification curves for pHi changes in DU145 cells after adding sodium acetate solution, following 30 min of ammonium chloride incubation.) The rectification ratio of pHi changes in DU145 cells before and after the addition of sodium acetate solution was detected using a pHi probe. Statistical graphs (n=6) are shown below. Figure 16 As shown, from Figure 16 As can be seen from the data, the rectification ratio of six independent DU-145 cells decreased after the addition of sodium acetate solution, indicating that the cell pHi decreased in the presence of sodium acetate. The cell pHi decreased from 7.42±0.17 to 7.08±0.10, which shows that the pHi probe prepared in this invention can monitor drug-induced pHi changes.

[0031] In summary, this invention discloses a method for preparing a pHi probe by inserting a nanoparticle into a cell. The pHi probe is obtained by incubating the nanoparticle inside the cell for 60 minutes. This preparation method is simple, efficient, and has low equipment dependence. The prepared nanoparticle-based pHi probe is sensitive to pH and can monitor the dynamic changes of single-cell pHi in real time. This preparation method and the resulting pHi probe overcome the technical limitations of traditional single-cell pH detection tools, such as cumbersome preparation processes, high equipment costs, and poor response time. It plays a significant role in promoting the innovative development and industrial application of single-cell analysis technology.

[0032] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a pHi probe based on live cell incubation, characterized in that, Includes the following steps: S1: The capillary tube is drawn into a nanotube with an inner diameter of 120 nm; S2: The nanopipette prepared in S1 was inserted into the cells and incubated for a period of time. Then, the nanopipette was rinsed with ultrapure water and then placed in a phosphate-free PBS solution for storage. After storage, the pHi probe based on live cell incubation was obtained.

2. The method for preparing a pHi probe based on live cell incubation according to claim 1, characterized in that, In S2, the incubation time is 10 min to 90 min.

3. The method for preparing a pHi probe based on live cell incubation according to claim 1, characterized in that, In S2, the cell types include human glioblastoma cells T98G, human renal epithelial cells 293T, human liver cancer cells HCCLM3, human prostate cancer cells DU-145, microglia BV2, and placental villous trophoblast cells HTR-8.

4. The method for preparing a pHi probe based on live cell incubation according to claim 1, characterized in that, In S1, the dimensions and material of the capillary are as follows: Borosilicate capillary tube BF100-59-10 with an inner diameter of 0.59 mm and an outer diameter of 1.00 mm; Borosilicate capillary tube BF120-69-15 with an inner diameter of 0.69 mm and an outer diameter of 1.20 mm; Borosilicate capillary tube BF100-78-15 with an inner diameter of 0.78 mm and an outer diameter of 1.00 mm; Borosilicate capillary tube BF150-86-10 with an inner diameter of 0.86 mm and an outer diameter of 1.50 mm; Quartz capillary tube BF100-70-7.5 with an inner diameter of 0.70 mm and an outer diameter of 1.00 mm.

5. The method for preparing a pHi probe based on live cell incubation according to claim 4, characterized in that, In S1, the parameters for drawing the capillary are as follows: Borosilicate capillary tube BF100-59-10: Heat=280, Fil=0, Vel=20, Del=145, Pull=150; Borosilicate capillary tube BF120-69-15: Heat=350, Fil=0, Vel=10, Del=145, Pull=160; Borosilicate capillary tube BF100-78-15: Heat=400, Fil=0, Vel=20, Del=145, Pull=180; Borosilicate capillary tube BF150-86-10: Heat=350, Fil=0, Vel=20, Del=145, Pull=160; Quartz capillary tube QF100-70-7.5: Heat=650, Fil=0, Vel=20, Del=145, Pull=220.

6. The method for preparing a pHi probe based on live cell incubation according to claim 1, characterized in that, The phosphate-removing PBS solution comprises 137 mM NaCl and 2.7 mM KCl, with a pH of 6.

82.

7. A pHi probe based on live cell incubation, characterized in that, The pHi probe based on live cell incubation is prepared by the method described in claim 1.

8. The application of the pHi probe based on live cell incubation as described in claim 7 in detecting intracellular pH.

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