A method for preparing a single-cell intracellular pH sensor based on nanopipette

Abstract

The present invention discloses a method for preparing a single-cell intracellular pH sensor based on a nanopipette, wherein a quartz capillary is drawn into a nanopipette with an inner diameter of 80 to 320 nm, a titanium trichloride solution is injected into the nanopipette, the nanopipette is inverted, and after standing in the dark, heating and reacting, titanium dioxide is generated at the tip of the nanopipette, the nanopipette is rinsed with ultrapure water and then placed in a buffer solution for storage. The present invention discloses a method for preparing a single-cell intracellular pH sensor based on a nanopipette, wherein a nanopipette intracellular pH sensor is prepared in the form of growing titanium dioxide at the tip of the nanopipette, an aqueous solution of titanium trichloride is injected into the tip of the nanopipette, and the nanopipette pH sensor can be prepared by heating. The method is simple, efficient, has low equipment dependence, few by-products, and has a high success rate and good repeatability. The prepared nanopipette pH sensor is sensitive to pH response and has high resolution, and can monitor the dynamic changes of single-cell intracellular pH in real time.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a method for preparing a single-cell intracellular pH sensor based on a nanostraw. Background Art

[0002] Cells are the basic units of life activities of organisms. Their intracellular pH plays an important role in protein function, cell metabolism, growth, proliferation, migration and other cell processes. Changes in intracellular pH are often a sign of pathological changes, such as carcinogenesis, apoptosis, myocardial ischemia, Alzheimer's disease, etc. At the same time, there are reports that exogenous compounds (such as drugs and alcohol) can change intracellular pH. Therefore, intracellular pH is an important biomarker in cancer research, neuroscience, metabolism, cell biology and pharmacology. The sensing of intracellular pH is of great significance to the study of cell pathophysiology, early diagnosis of diseases, drug screening and drug action kinetics.

[0003] In the past two decades, the development of several analytical techniques has made single-cell intracellular pH sensing possible, such as fluorescence-based spectroscopy / microscopy, plasma / surface-enhanced Raman spectroscopy, nanopore / nano-straw sensing, etc. Among them, the fluorescence-based pH fluorescent probe cannot provide reliable intracellular pH sensing with high spatial resolution due to problems such as uneven and uncontrollable distribution within the cell. The long pH response and data acquisition time of the plasma nanotip based on surface-enhanced Raman spectroscopy will lead to the problem of insufficient temporal resolution. Intracellular sensing based on nanopore / nano-straw has obvious advantages. It has the advantages of high electrochemical detection sensitivity, fast response speed, and real-time dynamic monitoring. At the same time, its nanoscale tip (less than 10nm) can penetrate into tiny areas (such as organelles) for sensing, so it can achieve high temporal and spatial resolution intracellular sensing of single cells.

[0004] Nanostraws are tubular objects with a hollow structure at the nanometer scale and a tip opening. They are usually made of glass capillaries through a microelectrode puller. Unlike traditional straws used to transfer liquids, nanostraws are widely used in single-cell experiments such as intracellular injection, cell biopsy, and intracellular sensing due to their nanometer-sized tips, hollow structures, and ion transport behaviors that can respond to external stimuli. The response of the ion transport behavior of the nanopores at the tip of the nanostraw to the target signal is the basis for its intracellular sensing. In order to achieve an effective response of the nanostraw to pH, researchers have introduced chemical groups into the inner wall of the nanostraw to enhance the pH response of the nanostraw. A series of achievements have been made in the nanopores of nanostraws, including but not limited to nanostraws: a variety of inner wall groups have been successfully introduced into the inner wall of the nanostraw, and the pH response of the nanostraw has been enhanced. Despite the promising prospects, the research and development of nanostraw pH sensors is still in its early stages, especially in the functional modification of nanostraws. There are still great challenges, such as insufficient sensitivity to pH response, insufficient resolution, low success rate, and poor repeatability. Summary of the invention

[0005] The present invention provides a method for preparing a single-cell intracellular pH sensor based on a nanopipette, so as to solve the problems of low sensitivity, insufficient resolution, low success rate and poor repeatability of the nanopipette pH sensor.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for preparing a single-cell intracellular pH sensor based on a nanopipette comprises the following steps:

[0008] S1: Pull the quartz capillary into a nanopipette with an inner diameter of 80-320nm;

[0009] S2: Inject the prepared titanium trichloride solution into the nanostraw obtained in step S1, then invert the nanostraw, keep it still in the dark, heat it for reaction, rinse the nanostraw with ultrapure water and store it in a buffer solution.

[0010] Furthermore, in step S1, the inner diameter of the quartz capillary is 0.7 mm and the outer diameter is 1.0 mm.

[0011] Further, in step S1, the drawing parameters are Heat=700, Fil=4, Vel=60, Del=170, Pull=70-180;

[0012] Within the allowable error range, when the drawing parameters are Heat = 700, Fil = 4, Vel = 60, Del = 170, Pull = 180, the inner diameter of the obtained nanostraw is 80nm; when the drawing parameters are Heat = 700, Fil = 4, Vel = 60, Del = 170, Pull = 150, the inner diameter of the obtained nanostraw is 130nm; when the drawing parameters are Heat = 700, Fil = 4, Vel = 60, Del = 170, Pull = 150, the inner diameter of the obtained nanostraw is 130nm. =60, Del=170, Pull=100, the inner diameter of the obtained nanostraw is 180nm; when the drawing parameters are Heat=700, Fil=4, Vel=60, Del=170, Pull=80, the inner diameter of the obtained nanostraw is 250nm; when the drawing parameters are Heat=700, Fil=4, Vel=60, Del=170, Pull=70, the inner diameter of the obtained nanostraw is 320nm.

[0013] Furthermore, in step S2, the concentration of the titanium trichloride solution is 5-100 mM.

[0014] Furthermore, in step S2, the amount of titanium trichloride solution added is 15-30uL.

[0015] Furthermore, in the step S2, the light-proof standing time is 10 to 120 minutes.

[0016] Furthermore, in step S2, the reaction temperature is 75-98° C. and the reaction time is 10-120 min.

[0017] Furthermore, in step S2, the buffer solution is a phosphate buffered saline solution.

[0018] Furthermore, the phosphate buffered saline solution is prepared into a solution with a pH value of 7.2 by 8 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl.

[0019] The invention discloses a method for preparing a single-cell intracellular pH sensor based on a nanopipette. The nanopipette intracellular pH sensor is prepared by a one-step reaction. An aqueous solution of titanium trichloride is injected into the tip of the nanopipette, and the nanopipette pH sensor can be prepared by heating. The method is simple, efficient, has low dependence on equipment, few by-products, high success rate, and good repeatability. The prepared nanopipette pH sensor is sensitive to pH response, has high resolution, and can monitor the dynamic changes of single-cell intracellular pH in real time. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is a light microscope image of the nano-straw before the reaction of the present invention;

[0022] Figure 2 This is a light microscope image of the nano-straw after the reaction of the present invention;

[0023] Figure 3 is the scanning electron microscope image of the nanostraw after the reaction;

[0024] Figure 4 The current response of the grown titanium dioxide nanostraw (180 nm) to pH;

[0025] Figure 5 This is the high-resolution current response of the titanium dioxide-grown nanopipette (180 nm) to pH. The dashed box in the inset is an enlarged view.

[0026] Figure 6 is the linear relationship between the ionic current and pH of the nanopipette grown on titanium dioxide (180 nm) (n=6);

[0027] Figure 7 Reversible response of ionic current to pH of titanium dioxide-grown nanopipette (180 nm) (n=6);

[0028] Figure 8 The sensitivity of the nanostraws with different diameters to pH response after the titanium dioxide was grown (n=6);

[0029] Fig. 9 is the sensitivity of the nanostraws to pH response after the growth of titanium dioxide at different titanium trichloride concentrations (n=6);

[0030] Fig.10 is the sensitivity of the nanostraws to pH response after titanium dioxide is grown at different temperatures (n=6);

[0031] Fig.11 The sensitivity of the nanostraws to pH response after titanium dioxide growth at different reaction times (n=6);

[0032] Fig.12 Microscopic image of a titanium dioxide-grown nanopipette piercing human breast cancer cells (MCF-7) for pH monitoring;

[0033] Fig.13To grow titanium dioxide nanostraws and monitor the intracellular pH changes of MCF-7 cells under the action of omeprazole. DETAILED DESCRIPTION

[0034] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-13 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Embodiment 1:

[0036] A quartz capillary with an outer diameter of 1.0 mm and an inner diameter of 0.7 mm was drawn into a nano-straw with an inner diameter of 180 nm using a P-2000 microelectrode laser drawing apparatus. The straightening parameters were Heat = 700, Fil = 4, Vel = 60, Del = 170, and Pull = 100.

[0037] Use ultrapure water to prepare a 50mM titanium trichloride solution, then use a micropipette to transfer 20uL of the titanium trichloride solution into a nanostraw with an inner diameter of 180nm. Then, invert the nanostraw and place it in a centrifuge tube, keep it away from light for 1 hour, and then put it in a 90℃ oven. After 1 hour, rinse the nanostraw thoroughly with ultrapure water and store it in a centrifuge tube containing phosphate buffered saline (PBS) with a pH value of 7.2.

[0038] Performance testing:

[0039] In order to circumvent the pH buffering capacity of PBS, the phosphate buffered saline solution in the lumen of the nanostraw was replaced with a saline solution (137mM NaCl, 2.7mM KCl). An Ag / AgCl reference electrode with a diameter of 0.35mm was inserted into the tail end of the nanostraw, and then the nanostraw and another Ag / AgCl reference electrode were simultaneously inserted into the PBS buffer of the electrolytic cell. The CHI 660A electrochemical workstation was used to connect the two reference electrodes to record the data generated during the experiment, and the ion current generated was recorded using the chronoamperometry (-1V). Among them, the initial electrolyte solution in the electrolytic cell was PBS with a pH value of 8, and the pH of the PBS solution in the electrolytic cell was adjusted with a phosphate buffer containing 137mM NaCl, 2.7mM KCl, and 10mM H3PO4.

[0040] Take the MCF-7 cells in the logarithmic growth phase and discard the culture medium. Rinse with 1×PBS for 3 times, digest with trypsin and add to 1×PBS. Use a microscope, micro-ultrasound system (MPC365) and electrochemical workstation (CHI 660A) to detect the intracellular ion current of single cells (the whole device is placed on an optical plate, and the air bag is placed under the optical plate to cushion the shock). Take 4.5mL of the culture medium containing MCF-7 cells (the number of cells is about 1×10 4 ), when the micron glass tube is close to the cell, slowly pull the syringe piston to create a low-pressure environment in the micron capillary cavity, and the cell is adsorbed to the front end of the micron glass tube. The right arm fixes the quartz nanopipette to puncture the cell. When adding drugs, slowly add 500μL of 1mM omeprazole (OM) to make the concentration of OM 100μM. The control group adds 500μL of dimethyl sulfoxide (DMSO) diluted in the same proportion without drugs. A silver / silver chloride electrode (0.35mm) is placed in the culture dish solution, and another silver / silver chloride electrode is inserted into the lumen from the rear end of the quartz nanopore. The ion current recorded by It (-1V) is used to monitor the changes in the intracellular pH of MCF-7 cells in the drug-added group and the control group.

[0041] like Figure 1-3 As shown, from Figure 1 As can be seen in Figure 2, no clogging was observed on the bare nanopipette tip. Figure 2 It can be seen from the figure that the titanium trichloride in the solution is heated to generate solid titanium dioxide, which blocks the tip of the nanostraw. After the titanium dioxide grows, obvious blockage can be observed at the tip of the nanostraw. Figure 3 It can be seen that the inner diameter of the tip of the nanostraw is about 180nm, and the outer diameter is 262nm. Titanium dioxide grows and fills the pores of the tip of the nanostraw, and some titanium dioxide is attached to the outer wall.

[0042] Figure 4 As can be seen from the figure, with the change of pH value, the nanostraws grown with titanium dioxide have a step-like current response. Different pH values ​​correspond to different current values. When the pH value of the solution decreases from 8 to 3, the response current increases from -11.75nA to -2.95nA in a step-like manner. This change value is 5.85 times the response current value at pH 3, indicating that the electrode has a sensitive response to pH.

[0043] Figure 5 As can be seen from the figure, the pH value of the solution was adjusted three times in succession to decrease by 0.05 each time, and the nanostraws grown on titanium dioxide all had a step-like current response, indicating that the nanostraws can effectively distinguish the difference of 0.05 in pH value and have a high resolution for pH sensing.

[0044] Figure 6In order to avoid the difference between the initial currents of the nanopipette, normalized data processing was performed; that is, the response current value of the nanopipette when the pH value of the solution was set to 8 was 100%, and the response current at other pH values ​​was compared with this current value to obtain a percentage value representing the response current value. It can be seen from the figure that within the pH range of 3 to 8, the normalized current value increases with the increase of pH value, and its sensitivity to pH response is 15.04% pH -1 Using the ANOVA test with a confidence level of 99%, p < 0.01, it was shown that there was a significant linear relationship between pH value and normalized current value. Figure 7 As can be seen from the figure, after repeated testing in acid and base solutions with pH values ​​of 4 and 8, the nanostraws showed good reversibility and repeatability of pH response. After repeated testing 8 times, the ion current still did not change significantly, indicating that the pH sensor has good stability.

[0045] Figures 8 to 10 The sensitivity of the nanostraw to pH response after growing titanium dioxide under different conditions; the change in the response current of the nanostraw is tested (when the pH value of the solution decreases from 8 to 3), and the change is compared with the response current value when the pH value is 8 to obtain the response current ratio, and the response current ratio is used to represent the sensitivity of the nanostraw to pH response.

[0046] Figure 8 As can be seen from the figure, nanopipette with inner diameters of 80±5nm, 130±5nm, 180±5nm, 250±5nm, and 320±5nm can all respond to pH after growing titanium dioxide. When the pH value of the solution drops from 8 to 3, the change ratio of the response current is 57.2%±0.022, 72.60%±0.017, 75.22%±0.017, 69.09%±0.014, and 67.99%±0.015, respectively. Among them, the nanopipette with an inner diameter of 180±5nm is the most sensitive to pH.

[0047] Fig. 9 As can be seen from the figure, with the increase of titanium trichloride concentration in the substrate, the pH response sensitivity of the nanostraw first increases and then decreases, and the optimization effect is best at 50mM.

[0048] Fig.10 As can be seen from the figure, with the increase of reaction time, the pH response sensitivity of the nanostraw first increases and then decreases slightly and remains stable. When the reaction time is above 10 minutes, a nanostraw pH sensor that effectively responds to pH can be successfully prepared.

[0049] Fig.11As can be seen from the figure, when the reaction temperature is above 75°C, the nanostraws have a significant response to pH, and 90°C is the optimal reaction temperature.

[0050] Fig.12 As can be seen from the figure, after the micron capillary adsorbs MCF-7 cells at low pressure, the nanopipette successfully penetrates into the cell for real-time sensing.

[0051] from Fig.13 It can be seen that in the control group without treatment, the induced current of the nanopipette remained stable, indicating that the intracellular pH of the cells remained stable; for cells that only added dimethyl sulfoxide (omeprazole solvent), the nanopipette pH sensor did not detect obvious current changes. The current disturbance at the addition point was considered to be the electric field interference caused by the addition of the solution, which caused the induced current fluctuations. After the liquid level stabilized, the induced current disappeared; for cells that added the proton pump inhibitor omeprazole, after 2 minutes, the proton pump was inhibited, resulting in cell acidification. Subsequently, due to the adaptability of the cells, the intracellular acidification level decreased, but it was still maintained and balanced at an acidified level. The nanopipette pH sensor clearly observed and recorded this process through the induced current.

[0052] The present invention discloses a method for preparing a single-cell intracellular pH sensor based on a nanostraw, wherein the nanostraw intracellular pH sensor is prepared by growing titanium dioxide at the tip of the nanostraw, and an aqueous solution of titanium trichloride is injected into the tip of the nanostraw, and the nanostraw pH sensor can be prepared by heating. The method is simple, efficient, has low dependence on equipment, has few by-products, and has a high success rate and good repeatability. The prepared nanostraw pH sensor is sensitive to pH response, has high resolution, and can monitor the dynamic changes of single-cell intracellular pH in real time.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 single-cell intracellular pH sensor based on a nanopipette, characterized in that: The following steps are involved: S1: Pull the quartz capillary into a nanopipette with an inner diameter of 180 nm; S2: injecting the prepared titanium trichloride solution into the nano-straw obtained in step S1, then inverting the nano-straw, keeping it still in the dark, heating it for reaction, rinsing the nano-straw with ultrapure water and storing it in a buffer solution; In the step S2, the concentration of the titanium trichloride solution is 50 mM; the amount of the titanium trichloride solution added is 20 uL; the light-proof standing time is 10 to 120 min; the reaction temperature is 75 to 98° C., and the reaction time is 10 to 120 min.

2. The method for preparing a single-cell intracellular pH sensor based on a nanostraw according to claim 1, characterized in that: In the step S1, the inner diameter of the quartz capillary is 0.7 mm and the outer diameter is 1.0 mm.

3. The method for preparing a single-cell intracellular pH sensor based on a nanostraw according to claim 1, characterized in that: In the step S1, the drawing parameters are Heat=700, Fil=4, Vel=60, Del=170, Pull=70-180.

4. The method for preparing a single-cell intracellular pH sensor based on a nanostraw according to claim 1, characterized in that: In step S2, the buffer solution is a phosphate buffered saline solution.

5. The method for preparing a single-cell intracellular pH sensor based on a nanostraw according to claim 4, characterized in that: The phosphate buffered saline solution is prepared into a solution with a pH value of 7.2 by 8mM Na2HPO4, 2mM KH2PO4, 137mM NaCl and 2.7mM KCl.

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