WO2022164262A1

WO2022164262A1 - Nanoprobe for measuring intracellular ph, and method and apparatus for measuring ph in single cell using same

Info

Publication number: WO2022164262A1
Application number: PCT/KR2022/001600
Authority: WO
Inventors: 제정호; 오승수; 용문중; 양운; 강병화
Original assignee: 포항공과대학교 산학협력단
Priority date: 2021-02-01
Filing date: 2022-01-28
Publication date: 2022-08-04
Also published as: KR102706187B1; KR20220111204A

Abstract

The present invention relates to a method and apparatus for measuring pH in a single cell, and a method for manufacturing a nanoprobe for same. The method for measuring pH in a single cell comprises (a) inserting, into the single cell, a nanoprobe formed by coupling a pH-responsive fluorescent material capable of responding to pH to a nanowire grown on a tapered tip of an optical fiber, (b) shining light on the nanoprobe through the optical fiber, (c) exciting the pH-responsive fluorescent substance by means of the light to generate fluorescence, (d) acquiring, through the optical fiber, a fluorescence signal generated from the fluorescent material according to the pH of the cell, and (e) obtaining the pH value in the cell by analyzing the fluorescence signal. The method for manufacturing a nanoprobe comprises (a) filling a nanopipette with a nanowire material solution and lowering the nanopipette to bring the nanowire material solution into contact with an end of an optical fiber, (b) raising the nanopipette to grow a nanowire on the end of the optical fiber, (c) filling a micropipette with an aqueous solution containing a pH-responsive fluorescent substance and lowering the micropipette so that a portion of the nanowire is immersed in the aqueous solution, and (d) raising the micropipette to form a nanoprobe labeled with the pH-responsive fluorescent substance.

Description

Nanoprobe for measuring intracellular PH and method and apparatus for measuring PH within a single cell using the same

The present invention relates to the measurement of pH in a single cell, and more particularly, to a nanoprobe capable of accurately measuring the pH in a single cell, and a method and apparatus for measuring the pH in a single cell using the same.

Conventionally, all cells were considered to be homogeneous and the cells were analyzed collectively, but it was recently found that individual cells are heterogeneous (see Cell Cycle 12, 3640-3649 (2013)). . Accordingly, recently, a technique for analyzing individual characteristics of a single cell has been attracting attention (see Nat. Cell Biol. 20, 1349-1360 (2018)).

Factors related to cellular properties are various such as pH, mRNA, and protein. Among them, pH is an important factor because it affects intracellular protein metabolism and directly affects cell function (see J. Immunol. Methods. 221, 43-57 (1998)). It is also known that intracellular pH measurement is utilized as a criterion for diagnosing diseases such as cancer (see Biochemistry . 35, 2811-2817 (1996)).

In particular, the nucleus of cancer cells divides rapidly and the pH is expected to be different from that of normal cells (see Chem. Soc. Rev. 46, 3830-3852 (2017); and Nat. Rev. Cancer . 11, 671677 (2011)). . However, it is known that it is very difficult to measure the pH inside the nucleus because the cell nucleus is not only deep in the cytoplasm but also surrounded by a nuclear membrane. Therefore, measuring the pH in a single cell nucleus is a more difficult technique than the pH of a single cytoplasm.

For this reason, conventionally, the following methods have been used to measure the pH in a single cell.

First, a nanoparticle insertion-based technology for measuring intracellular pH measures pH by inserting a fluorescent nanomaterial that responds to pH into the cell, and then analyzing the signal outside the cell ( J. Am. Chem. See Soc. 136, 12253-12256 (2014); Anal. Chem. 91, 8383-8389 (2019); and Analyst 145, 5768-5775 (2020)). However, analysis of cells in their natural state is impossible due to the insertion of foreign substances into cells, and single cell analysis is very difficult due to the randomness of the insertion of fluorescent nanomaterials into cells. In addition, since the intracellular signal is measured outside the cell, light scattering is inevitable, so the accuracy of pH measurement is poor. Moreover, it is known that it is very difficult to measure the pH in the nucleus because it is almost impossible to insert nanomaterials into the nucleus of a single cell.

Another method is a probe insertion-based pH measurement method, and there is a technology for measuring intracellular pH by inserting a probe containing a substance that responds to pH into the cell ( Sensors Actuators, B Chem. 290, 527-). 534 (2019); and Analyst 145, 4852-4859 (2020)).In this method, a probe is prepared by binding a pH-responsive material to the surface of a tapered glass capillary. Therefore, the diameter of the probe becomes thicker as it goes away from the tip of the probe, and when inserted to a desired position in the cell, the cell may be damaged. Then, the laser is irradiated from the outside of the cell towards the probe inside the cell, and the reflected light is again detected outside the cell. will measure Here, since light is irradiated from the outside of the cell and the reflected light is received from outside the cell, severe scattering of light is unavoidable in the process of light passing through various media, resulting in poor accuracy.

Accordingly, the present invention has been developed to solve the above problems, and a nanoprobe capable of accurately measuring the pH in a single living cell in real time without contamination or damage to the cell during intracellular insertion, and a method for measuring intracellular pH using the same and to provide an apparatus.

According to one aspect of the present invention for achieving the above object, the method for measuring the pH in a single cell is (a) pH responsiveness that can respond to pH in a nanowire grown on the tapered tip of an optical fiber inserting a nanoprobe formed by binding a fluorescent material into a single cell; (b) incident light to the nanoprobe through the optical fiber; (c) excitation of the pH-responsive fluorescent material by the light to generate fluorescence; (d) acquiring a fluorescent signal generated from the fluorescent material through the optical fiber according to the pH of the cell; and (e) analyzing the fluorescence signal to obtain a pH value in the cell.

The method for manufacturing a nanoprobe according to the present invention comprises: (a) filling a nanopipette with a nanowire material solution and lowering the nanopipette to bring the nanowire material solution into contact with the tip of an optical fiber; (b) raising the nanopipette to grow a nanowire on the end of the optical fiber; (c) filling a micropipette with an aqueous solution containing a pH-responsive fluorescent substance and lowering the micropipette so that a part of the nanowire is immersed in the aqueous solution; and (d) raising the micropipette to form a nanoprobe labeled with a pH-responsive fluorescent material. a nanowire formed by growing a nanowire material solution at one end of the optical fiber; and a pH-responsive fluorescent material labeled on a part of the nanowire.

In the present invention, the nanowire material solution is a hydrophobic polymer solution, and at least PVBN ₃ , PVB-alkyne, and PVB-COOH may be selected from the group consisting of. In addition, the optical fiber has a tapered tip, and the pH-responsive fluorescent material is a fluorescein molecule having a functional group capable of binding to the nanowire - wherein the fluorescein is at least DBCO-FAM, Azide-FAM, or Amine-FAM may be selected from the group consisting of -. According to the present invention, the wetting (or labeled) length of the nanowire by the pH-responsive fluorescent material is controlled to be 100 nm to 900 nm, preferably 500 nm or less.

As another aspect of the present invention, an apparatus for measuring pH in a single cell includes: a nanoprobe formed by combining a pH-responsive fluorescent material capable of responding to pH to a nanowire grown on a tapered tip of an optical fiber; a manipulator capable of three-dimensional movement control of the nanoprobe to insert the nanoprobe into the single living cell; a light source for applying light to the optical fiber; an optical coupler connecting the optical fibers to transmit the light incident through the optical fiber to the nanoprobe and to transmit the fluorescence signal generated from the nanoprobe to the spectrometer through an additional optical fiber; and a spectrometer to obtain a pH value by analyzing spectral data from the fluorescence signal generated by the nanoprobe.

In the present invention, the nanoprobe may have a uniform diameter. The nanoprobe has a diameter of 10 nm to 900 nm, preferably 400 nm or less, and a length of 1 μm to 10 μm, preferably 5 μm or less.

In the present invention, the light incident through the optical fiber may be near-infrared or visible light, and the light may have a wavelength of 300 nm to 1000 nm, preferably 400 nm to 700 nm. The wavelength of light incident through the optical fiber is selectable according to the component, shape and optical properties of the nanoprobe, the type of target molecule to be detected, and the type of target cell.

As another aspect of the present invention, the method for preparing the nanowire material solution is a mixture of PVC (0.014 g, 131 mmol) and sodium azide (0.010 g, 220 mmol) in anhydrous DMF solvent (0.7 mL). Mix in an amber vial at 70 °C and cover with aluminum foil to block light; After 2 hours of reaction, methanol (0.5 mL) is added, and the mixed solution is centrifuged at 10,000 rpm for 1 minute to remove excess unreacted reagent and precipitate an azide-functionalized polymer; and drying the obtained precipitate under vacuum for 1 hour and then dissolving it by adding an NMP solvent (50 μL).

Although it is important to understand cellular heterogeneity and metabolism through local pH monitoring, spatio-temporal pH monitoring of single living cells beyond cell and organelle membranes is challenging.

In the present invention, the inventors have developed a nanoprobe with high mechanical strength that enables in situ monitoring of pH dynamics in desired organelles through direct optical communication. By chemically labeling one end of polyvinylbenzyl azide nanowire with fluorescein, pH changes in different compartments inside living cells are continuously monitored to adjust pH homeostasis of specific organelles and pH according to stimuli. was successfully observed.

Importantly, it was demonstrated for the first time that during the human cell cycle, the nucleus exhibits pH homeostasis in the resting phase but changes in pH during the mitotic phase, thereby participating in independent pH regulation by the nuclear membrane. The fast and accurate local pH detection and reporting function of nanoprobes is very useful for investigating cellular behavior in various biological situations of various living cells.

On the other hand, according to the above-described features, the present invention provides the following effects.

1) If the device for measuring the pH in a single cell using the nanoprobe according to the present invention is used, the pH can be accurately measured for each position inside the single cell without contamination and damage to the cell when inserted into the cell.

2) Using the pH measuring device according to the present invention, it is possible to measure the change in pH according to time or environment in a single cell in real time without contamination and damage to the cell when inserted into the cell.

3) Using the pH measuring device according to the present invention, it is possible to accurately measure the pH of the cytoplasm and cell nucleus of a single cell without contamination or damage to the cell when inserted into the cell, and the pH in other organelles in the cell Of course, it can also be accurately measured.

4) Using the pH measuring device according to the present invention, it is possible to measure the pH change in the nucleus during the growth of a single cell in real time.

1 is a device configuration diagram (a) of a nanoprobe capable of detecting and transmitting a change in pH over time in a single living cell (a), an electron micrograph (b) of the nanoprobe, and pH detection in a single living cell using the nanoprobe. A drawing showing (c),

Figure 2 is a graph showing the 1H-NMR spectrum of PVC (Poly (vinylbenzyl chloride)) and PVBN3 (Poly (vinylbenzyl azide)),

3 is a view and a photograph showing the steps of making a PVBN ₃ nanowire at the end of a tapered optical fiber;

Figure 4 is a photograph showing the binding step of DBCO functionalized fluorescein (FAM) with PVBN ₃ nanowires,

5 is a photograph showing the evaluation of the mechanical properties of the nanoprobe inserted into the agar gel;

6 is a photograph showing the evaluation of light loss at the junction of the nanowire and the tapered optical fiber;

7 is a graph and a photograph showing the optical response of the nanoprobe to the local pH change;

8 is a graph of the light stability and reproducibility test of the nanoprobe;

9 is a photograph comparing the cell viability between the nanoprobe and the tapered optical fiber insertion into living HeLa cells;

10 is a graph showing a histogram of cell viability after the inserted nanoprobe (grey) or tapered optical fiber (white) was extracted from the cytoplasm and nucleus, respectively;

11 is a diagram illustrating a Boltzmann fitting to obtain a pH calibration curve targeting the intracellular environment;

12 is a photograph and graph showing the results of investigation of pH-dependent fluorescence signals of nanoprobes outside and inside HeLa living cells;

13 is an image and graph showing the pH value monitoring results in the cytoplasm and the cell nucleus during the entire cycle of a single cell using a nanoprobe;

14 is a photograph of brightfield images and merged (brightfield and fluorescence) images observed by confocal microscopy, showing the insertion of nanoprobes into single HeLa living cells during mitosis;

Figure 15 is a confocal microscopically observed merged (bright field and fluorescence) image photograph of HeLa cells stained with a nuclear-specific Hex dye (white) during mitosis (from electrophoresis to cytoplasmic division) for pH measurement;

16 is a diagram and a photograph showing changes in cytoplasmic pH in response to external calcium ions;

17 is a photograph and graph showing the real-time measurement result of the cytoplasmic pH of HeLa cells treated with an excess of magnesium ions (5 mM);

18 is a merged (bright-field and fluorescence) image and dark-field image photograph of living HeLa cells treated with excess calcium ions (a) and excess magnesium ions (b).

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. In the following embodiments, the illustration and description will be omitted except for essential parts in describing the invention.

Cells are different. Even in the same environment, genetically identical cells can exhibit cell-to-cell variabilities (eg, cell morphology, proliferation, growth and viability) due to individual compartmentalization (Stoeger, T., Battich, N. & Pelkmans, L. Passive Noise Filtering by Cellular Compartmentalization. Cell 164, 1151-1161 (2016)). To understand the various behaviors of individual cells, it is important to measure and analyze changes in physiological parameters (eg, pH, temperature, and oxygen levels) inside living cells (Zhang, X. ai et al. Quadruply-labeled serum albumin as a biodegradable nanosensor for simultaneous fluorescence imaging of intracellular pH values, oxygen and temperature (see Microchim. Acta 186, (2019)). In particular, since organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus perform biological functions from time to time, changes in different organelles must be individually monitored over time (Jaworska, A., Malek, K. & Kudelski, A. Intracellular pH - Advantages and pitfalls of surface-enhanced Raman scattering and fluorescence microscopy - A review. Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 251, 119410 (2021), and Han, J. & Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 110, 2709-2728 (2010)). In particular, due to different levels of cellular metabolism and homeostasis, there may be temporal and temporal pH heterogeneity between cells (Søndergaard, RV, Henriksen, JR & Andresen, TL Design, calibration and application of broad-range optical nanosensors for determining intracellular). see pH. Nat. Protoc. 9, 2841-2858 (2014)). Theoretically, local pH was predicted to fluctuate during cell division by successive catabolism or anabolic processes, and when activated by apoptotic stimuli, programmed cell death results in mitochondrial dysfunction, Leads to rapid acidification of the intracellular environment (Matsuyama, S., Llopis, J., Deveraux, QL, Tsien, RY & Reed, JC Changes in intramitochondrial and cytosolic pH: Early events that modulate caspase activation during apoptosis. Nat. Cell Biol 2, 318-325 (2000)) .

Due to the importance of local pH changes, extensive research has been conducted on the development of systems that can detect and transmit intracellular pH in real time. A variety of pH-responsive molecular probes (e.g., fluorescent dyes, quantum dots and nanoparticles) can penetrate impermeable cell membranes and enter cells via electroporation or endocytosis and can be used for pH detection (He, C., Lu, K. & Lin, W. Nanoscale metal-organic frameworks for real-time intracellular pH sensing in live cells. J. Am. Chem. Soc. 136, 12253-12256 (2014); D., Dublin, SN & Bao, G. Quantum dot-fluorescent protein fret probes for sensing intracellular pH. ACS Nano 6, 2917-2924 (2012); Shen, Y. et al. Organelle-targeting surface-enhanced Raman scattering ( SERS) nanosensors for subcellular pH sensing.Nanoscale 10, 1622-1630 (2018); and Albertazzi, L., Storti, B., Marchetti, L. & Beltram, F. Delivery and subcellular targeting of dendrimer-based fluorescent pH sensors in living cells. J. Am. Chem. Soc. 132, 18158-18167 (2010)). However, due to the nature of spontaneous intracellular insertion, placing the probe in a desired location, especially inside a membrane-enclosed organelle, still remains a technical challenge. Although pH-responsive fluorescent proteins can be genetically encoded inside engineered cells, their subsequent transport by sophisticated genetic engineering and protein trafficking associated with the expression of pH-responsive fluorescent proteins is very difficult. (Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys. J. 74, 1591-1599 (1998); and Palmer, AE, Qin, Y., Park, JG & McCombs, JE Design and application of genetically encoded biosensors. Trends See Biotechnol. 29, 144-152 (2011)). Alternatively, nanopipettes (Zhang, Y. et al. Spearhead Nanometric Field-Effect Transistor Sensors for Single-Cell Analysis. ACS Nano 10, 3214-3221 (2016); and Guo, J. et al. D ynamic single-cell) intracellular pH sensing using a SERS-active nanopipette. See Analyst 145, 4852-4859 (2020)) or optical fiber (Yang, Q. et al. Label-free in situ pH monitoring in a single living cell using an optical nanoprobe. Med. Devices Sensors 3, 1-10 (2020)) were directly inserted into target cells. However, unless the size and shape of the probe is precisely controlled, puncturing the membrane is lethal to the cells. In addition, the pH detection could not be localized due to the difficulty of surface modification and manipulation of the nanostructured material, and the weak detection signal was easily distorted by the complex cellular environment (Yan, R. et al. Nanowire-based single-cell endoscopy. Nat. Nanotechnol. 7, 191-196 (2011); and Lin, L. et al. Real-time tracing the changes in the intracellular pH value during apoptosis by near-infrared ratiometric fluorescence imaging. Chem. Commun. 54, 9071- 9074 (2018)). Therefore, a technique that enables real-time pH monitoring by intracellular organelles across multiple impermeable membranes of a single living cell is still in need of development.

In the present invention, the present inventors fabricated a nanoprobe with high mechanical strength and sufficiently small diameter to monitor the pH change of a desired cell compartment through direct optical communication. The polyvinyl benzyl azide (PVBN ₃ ) nanowire according to the present invention is structurally strong and long enough to penetrate cell membranes and organelle membranes, but cell damage or leakage is negligible because of its small diameter (200 nm or less). there are very few High-density fluorescein, chemically labeled at the tip of the nanoprobe, can respond rapidly to local pH changes, and through the nanoprobe, a pH-responsive photoluminescence (PL) signal can be sent directly to the spectrometer. transmitted (<100 ms), minimizing signal distortion due to optical loss and ambient noise. Using the in-situ pH detection system according to the present invention, pH homeostasis of specific organelles and pH adjustment according to stimulation by continuously monitoring changes in the pH of various compartments inside a single living cell Several scientific information such as (stimuli-selective pH regulations) were obtained. In particular, during the cell cycle, the nucleus exhibits pH homeostasis in resting phase but changes in pH during mitosis, demonstrating for the first time that the nuclear membrane is independently involved in pH regulation. This is due to the unique ability of the nanoprobe of the present invention to enable live streaming of subcellular events with local pH monitoring of single living cells.

In the present invention, single intracellular pH measurement starts with forming a nanoprobe having a uniform diameter capable of responding to pH by directly growing it on the tip of a tapered optical fiber. Such a nanoprobe includes a pH-responsive fluorescent material on its surface, and a method of forming the nanoprobe will be described with reference to FIG. 3 in the Examples below. For reference, here, 'single cell' means to include a single living cell and a single dead cell. In addition, 'a single intracellular pH measurement' means to include not only the cell nucleus, but also the cytoplasm and pH measurement of other intracellular organelles.

The single intracellular pH measurement method of the present invention can accurately measure the pH inside a cell by inserting a nanoprobe into the cell and directly analyzing the fluorescence signal generated according to the pH of the cell through an optical fiber.

In addition, the single intracellular pH measuring method of the present invention can measure the intracellular pH change in real time by directly measuring the change in the fluorescence signal according to the change in the pH of the cell according to time or environment.

In addition, the single intracellular pH measurement method of the present invention has a sufficiently small and uniform diameter of the nanoprobe, so there is almost no cell damage, and by receiving a signal directly from a desired position in the cell, it is possible to accurately measure the pH at each intracellular location.

[Example]

1 shows the design configuration of the structure of a nanoprobe capable of detecting a change in pH with time and space in a single living cell and the pH detection in a single living cell using the nanoprobe. An overall schematic diagram of the nanoprobe system for in situ pH monitoring of compartments.

A nanoprobe (Nanoprobe or Nanowire waveguide; 1) combines a pH-responsive fluorescent material capable of responding to pH to a nanowire grown on the tapered tip 3 of the optical fiber 2 (see FIG. 1B) (FIG. 4A). to 4g), the laser incident from the light source (laser generator; 4) to the first optical fiber 2a reaches the nanoprobe 1 through the optical fiber 2 (white arrow).

In this embodiment, the optical fiber 2 includes a first optical fiber 2a for transmitting light (eg, a laser beam) incident from the light source 4 to the nanoprobe 1 and fluorescence generated from the fluorescent material on the surface of the nanoprobe. It is branched into a second optical fiber 2b for transmitting a signal to the spectrometer 8, and the first optical fiber 2a and the second optical fiber 2b are combined into one through a fiber coupler 5, and the nanoprobe It leads to (1). The optical coupler 5 transmits the light incident to the first optical fiber 2a only to the nanoprobe 1 and transmits the fluorescence signal generated from the fluorescent material on the surface of the nanoprobe 1 only to the spectrometer 8 . guide the progress.

The nanoprobe 1 is inserted into a single living cell 7 using a manipulator 6 capable of three-dimensional movement control (see Fig. 1c). The positioning of the nanoprobe in the living cell can be precisely controlled with a 3-axis micromanipulator 6 while observing with confocal fluorescence microscopy. At this time, the light (laser beam) reaching the nanoprobe 1 from the light source 4 through the first optical fiber 2a and the optical coupler 5 generates an evanescent wave, and the pH reactivity at the tip of the nanoprobe By exciting the fluorescent material, the pH-responsive fluorescent material emits a photoluminescence (PL) signal. After this signal is transmitted to the optical fiber 2 through the nanoprobe 1, it is guided to the second optical fiber 2b by the optical coupler 5 without being subjected to environmental interference in optical communication, and directly to the spectrometer 8 transmitted (black arrow). Through this process, the pH value is measured from the spectral data of fluorescence obtained by the spectrometer 8. In this case, the fluorescence signal of the nanoprobe 1 from the inside of the cell (see FIG. 1c) is directly transferred to the spectrometer 8 without distortion. Because it is measured, it is possible to measure the accurate pH value.

Fig. 1b shows a field emission scanning electron microscope image (scale bar 1 μm) of a nanoprobe of the present invention grown directly on the tip of a tapered optical fiber, and Fig. 1c shows local pH monitoring of living cells across the rigid membrane of a cell or organelle. represents an image that The long and thin nanoprobe of the present invention is mechanically robust and does not induce cell leakage during membrane penetration, and the fluorescently labeled tip can easily reach a desired location (in the cytoplasm or nucleus) for in situ pH detection (inset). Reference). Depending on the local intracellular proton concentration, the intensity of the PL signal changes rapidly, which can be monitored in real time through a nanoprobe.

In the present invention, the nanoprobe 1 is capable of penetrating not only the cell membrane but also the nuclear membrane, so it is possible to measure the pH in the nucleus as well as the cytoplasm (see FIG. 1c; of course, it is also possible to measure the pH of other intracellular organelles). In addition, since the nanoprobe 1 has a sufficiently small diameter (d; 10 nm to 900 nm, preferably 400 nm or less), there is an advantage that there is no cell damage. In addition, since the nanoprobe 1 has a sufficiently small length ( l ; 1 μm to 10 μm, preferably 5 μm or less), it can be positioned at a specific location within the cell to measure pH at a desired location (cytoplasm, cell nucleus, etc.). In addition, since the pH-responsive fluorescent material on the surface of the nanoprobe 1 is in instantaneous chemical equilibrium with the proton, it is possible to accurately measure the change in the pH value in real time according to the change of time or environment in the cell.

In the present invention, the light source incident through the optical fiber 2a is a laser or LED, and may be light in the near-infrared or visible region, preferably light of a wavelength of 300 nm to 1000 nm, more preferably of a wavelength of 400 nm to 700 nm. can be light. However, the usable wavelength of light is not limited thereto, and may be arbitrarily selected according to the component, shape, and optical properties of the nanoprobe (optical nanowaveguide), the type of target molecule to be detected, the type of target cell, and the like.

A pH-responsive fluorescent dye at one end of a polymeric nanowire (Alvarez-Pez, JM, Ballesteros, L., Talavera, E. & Yguerabide, J. Fluorescein excited-state proton exchange reactions: Nanosecond emission kinetics and correlation with steady-state fluorescence intensity. J. Phys. Chem. A 105, 6320-6332 (2001)) - Here, the pH-responsive fluorescent dye is composed of fluorescein (DBCO-FAM, Azide-FAM, Amine-FAM, etc.) having a functional group capable of binding to the nanowire. By chemically labeling a molecule (selectable from the group), a nanoprobe suitable for in situ monitoring of local pH over time in single living cells was successfully prepared (Fig. 1a). Specifically, by evaporation of polyvinylbenzylazide (PVBN ₃ ) solution (M _n 52,000 g/mol) (FIG. 2), PVBN ₃ nanowires were directly grown on the tip of the tapered optical fiber (FIG. 1b and FIG. 1b and FIG. 3), the optical fiber was connected to the laser source and the spectrometer by a 1x2 fiber coupler (see “Method” below). Since the surface of the nanowire is filled with azide moieties (-N ₃ ), for example, by binding with dibenzocyclooctyne (DBCO) functionalized fluorescein (FAM), the tip (length of 100 nm to 900 nm, preferably 500 nm or less) could be selectively converted into high-density pH reporters through a copper-free click reaction (Fig. 4). Importantly, the nanoprobe of the present invention served as an excellent bidirectional optical signal transmission path for the PL signal from the excitation laser (white arrow) and localized fluorescein (black arrow). As the PL signal was transmitted directly to the spectrometer, the intensity of the PL spectrum, which changed according to the concentration of protons at a desired location, regardless of the environment around the nanoprobe, was measured in real time (see FIG. 1 ).

The physical and optical properties of the nanoprobe according to the present invention were very suitable for detecting and delivering intracellular pH inside living cells. Previous studies on nanowire diameters that minimize cell damage (Obataya, I., Nakamura, C., Han, SW, Nakamura, N. & Miyake, J. Direct insertion of proteins into a living cell using an atomic force microscope with Based on a nanoneedle. Nanobiotechnology 1, 347-352 (2005)), the Confined-growth method of the present invention (Je, JH; Yang, U.; Oh, SS; Yong, MJ; Kang, BH Method of forming micro) -or nanowires at predetermined positions of an object using a micro- or nanopipette, see US Patent 17,306,220, May 3, 2021) to prepare nanoprobes with a diameter of 200 nm or less ( FIG. 1b ). Despite their small diameter, due to the high modulus of elasticity of PVBN ₃ (E - 1.7 GPa) (see Bicerano, J. Prediction of Polymer Properties, 2nd ed . (Marcel Dekker, New York, 1996)), the nanoprobe has a rigid membrane. could easily penetrate; Higher modulus of elasticity (> 0.1 GPa) than actual cell membrane (E - 0.05 GPa) (Wang, K. et al. Specific membrane capacitance, cytoplasm conductivity and instantaneous Young's modulus of single tumour cells. Sci. Data 4, 1-8 (2017) ))), when the nanoprobe was inserted into the agar gel, no structural deformation was observed (see FIG. 5 ). Importantly, PVBN higher than the refractive index of the cellular environment (below 1.37) (see Liu, PY et al. Cell refractive index for cell biology and disease diagnosis: Past, present and future. Lab Chip 16, 634-644 (2016)) Refractive index of ₃ (below 1.67) (see kJames, J., Hanna, JM & Subila, KB Refractive Index Engineering using Polymer Nanocomposites . (PhD thesis, University of South Brittany, France, 2019)), and nanoprobe and tapered fiber The unique properties of the nanoprobe, including the smooth bonding between them, make it suitable for in situ and real-time pH monitoring. Since the scattering at the junction is negligibly very small (Fig. 6), the local fluorescence signal of fluorescein was easily collected and transmitted with an efficiency greater than 84% (Lee, J. et al. Quantitative Probing of Cu ²⁺ ). Ions Naturally Present in Single Living Cells. Adv. Mater. 28, 4071-4076 (2016)).

Figure 2 shows 1H-NMR spectrum of PVC (Poly (vinylbenzyl chloride)) and PVBN ₃ (Poly (vinylbenzyl azide)).

Top panel: 1H spectrum of PVC in DMSO-d ₆ . These include 7.30-6.00 ppm of aromatic rings (b), 4.81-4.38 ppm of —CH ₂ Cl(c) and 1.87-1.04 ppm of methylene of the PVC backbone (a). Lower panel: 1H spectrum of PVBN ₃ of DMSO-d ₆ . These include 7.30-6.00 ppm of aromatic rings (b), 4.35-3.80 ppm of —CH ₂ N ₃ (c′) and 1.87-1.04 ppm of methylene (a). The shift from 4.5 ppm (PVC, c) to 4.2 ppm (PVBN ₃ , c′) of —CH ₂ indicates that the chloride was substituted with azide, indicating that the synthesis of PVBN ₃ was successful. All 1H-NMR spectra were recorded at 500 MHz using DMSO-d ₆ as solvent at room temperature. Chemical shifts of all H-NMR spectra are based on the residual signal (δ2.50) of DMSO-d ₆ by BRUKER AVANCE Ascend 500.

3 shows the steps of making a PVBN ₃ nanowire at the end of a tapered optical fiber, and FIG. 3 a is a method of growing PVBN ₃ nanowires at the tip of a tapered optical fiber using the apparatus manufactured by the applicant directly for manufacturing the nanowire. The process is schematically shown.

FIG. 3b is an enlarged view of PVBN ₃ nanowires grown on the tip of a tapered optical fiber, and the growth process of these PVBN ₃ nanowires will be described in detail with reference to FIGS. 3c to 3f . First, according to FIG. 3C , a nanowire material solution, that is, a PVBN ₃ solution, is filled in a glass nanopipette, and the nanopipette is vertically lowered to contact the tip of the tapered optical fiber. In the present invention, the nanowire material solution is preferably a hydrophobic polymer solution (eg, PVBN3 , PVB-alkyne, PVB-COOH, etc. can be selected from the group consisting of).

According to Figure 3d, the tip of the nanopipette is in contact with the tip of the tapered optical fiber. According to FIG. 3E, when the nanopipette is pulled up vertically, the _PVBN3 nanowires grown on the ends of the tapered optical fibers are formed as the solvent of the PVBN3 solution evaporates. Figure 3f shows freestanding PVBN ₃ nanowires grown on the tip of a tapered optical fiber (scale bar, 10 μm).

Figure 4 shows the binding process of DBCO-functionalized fluorescein (FAM) to PVBN ₃ nanowires. According to Figure 4a, an aqueous solution (100 nM) containing DBCO-FAM molecules, a pH-responsive fluorescent dye, is detailed. A method of bonding (bonding) with the surface of PVBN ₃ nanowires grown on the ends of the tapered optical fibers according to FIG. 3 is schematically shown. More specifically, according to FIG. 4b, with the glass micropipette filled with an aqueous solution (100 nM) containing DBCO-FAM molecules, the glass micropipette is vertically lowered toward the PVBN ₃ nanowire, as shown in FIG. 4c . Immerse the tip of the wire into the aqueous solution in a glass micropipette. In Figure 4c, the DBCO-FAM molecule is a copper-free click reaction (Campbell-Verduyn, LS et al. Strain-promoted copper-free 'click' chemistry for 18F radiolabeling of bombesin. Angew. Chemie - Int. Ed 50 , 11117-11120 (2011)) bonded (bonded) to the azide group of PVBN ₃ nanowires. When the pipette is raised in this state, as shown in FIG. 4d , a FAM-labeled nanoprobe is formed (scale bar, 10 μm). Fig. 4e shows a bright field image of a nanoprobe grown on a tapered optical fiber obtained by confocal microscopy, Fig. 4f shows a dark field image, and Fig. 4g shows a merged image, respectively. In the dark field image (Fig. 4f), a green fluorescence signal (shown in white in the figure) is clearly observed at the tip of the nanoprobe, but no fluorescence signal is detected at the rest of the nanowire and the tapered fiber surface. The tip fluorescence is controlled to a length of 100 nm to 900 nm, preferably 500 nm or less, by precisely adjusting the wetting depth of the nanowire using a high-precision xyz motor stage with a position accuracy of 250 nm.

FIG. 5 shows the evaluation of the mechanical properties of the nanoprobe inserted into the agar gel. According to FIGS. 5A to 5C, bright field images before (a), in the middle (b) and after (c) of the insertion process are shown. Here, the white dotted line indicates the surface of the agar gel. It can be seen from this analysis that there is little deformation of the nanoprobe after insertion (scale bar, 10 μm).

6 shows the evaluation of light loss at the junction of the nanowire and the tapered optical fiber. According to FIGS. 6a to 6b, the bright field image ( FIG. 6a ) and the dark field image ( FIG. 6b ) of the nanowire guided laser light (473 nm) are is shown Light scattering is observed at the tip of the nanoprobe (bottom dashed circle), whereas there is almost no light scattering at the bonding site (upper dashed circle) (scale bar, 10 μm).

7 shows the optical response of the nanoprobe to the local pH change. The nanoprobe can measure a rapid change in pH, cause negligible damage to cells during insertion, and selectively respond to pH even in a complex intracellular environment. Indicate the characteristics of the probe.

According to FIG. 7a, when the pH of the droplet was changed from 4 to 8 (see inset), the immersed nanoprobe successfully exhibited a change in the PL spectrum according to pH upon laser (λ = 473 nm) excitation. According to Figure 7b, the time-dependent fluorescence signal (λ = 535 nm) was monitored in real time by the nanoprobe due to the rapid response (<100 ms) to the pH change. The black and gray arrows indicate the injection points of acidic and basic solutions, respectively. According to Fig. 7c, the nanoprobe (dotted arrow, diameter less than 200 nm) could be easily inserted into living cells (top), whereas the tapered optical fiber (solid arrow, tip diameter less than 200 nm) induced severe cell damage and leakage (bottom). ). To determine whether or not the cells are viable, HeLa cells were stained with calcein-AM (green) and propidium iodide (red) (scale bar 10 μm). According to FIG. 7D, after measuring the normalized PL peak intensity (I535/I685) in nigericin-treated cells in the pH range of 5 to 9 (n=3), the Boltzmann function (R ² = 0.9969) ) to obtain a pH calibration curve (black). As measured in HeLa cells treated with nigericin at pH 7.5 (n=3), the normalized PL peak intensities inside the cell (grey) and outside (white) are the same, so the intracellular and extracellular pH values are the same is shown (see inset).

The pH reactivity of the nanoprobes in various solutions with pH values ranging from 4 to 8 was investigated using a micro-photoluminescence system (Fig. 1a) (Fig. 7a). When the nanoprobe was immersed in droplets of different pH (buffer, 5 μl), pH-dependent PL spectra were successfully obtained upon laser excitation (λ = 473 nm), and the PL peak intensity at 535 nm (I ₅₃₅ ) was fluorescein (fluorescein). ) of the well-known pH-dependent properties (Alvarez-Pez, JM, Ballesteros, L., Talavera, E. & Yguerabide, J. Fluorescein excited-state proton exchange reactions: Nanosecond emission kinetics and correlation with steady-state fluorescence intensity. J Phys. Chem. A 105, 6320-6332 (2001)) showed a gradual increase with increasing pH. The PL intensity at 685 nm (I ₆₈₅ ) exhibited a negligible change with increasing pH, so it was used as a reference signal for the rest of the pH monitoring. Importantly, the nanoprobe exhibited good photostability and reproducibility in fluorescence detection, with negligible changes in PL peak intensity observed for 18 s of continuous laser exposure (Fig. 8a), and periodic changes in pH 5.0 to 7.5 PL peak intensity was reversibly changed during (Fig. 8b).

The PL spectrum through the nanoprobe responded to the pH change within a very short time (<100 ms) (Fig. 7b). For example, when a droplet immersed in a nanowire of pH 7.5 was rapidly changed (acidified) to pH 6.8 (Fig. 7b, black arrow), the PL peak intensity rapidly decreased within 100 ms. Conversely, when these weakly acidic droplets were rapidly mixed with a basic buffer solution, the PL peak intensity rapidly increased, showing a final pH of 7.2 (Fig. 7b, gray arrow). Fluorescein excited-state proton exchange reactions: Nanosecond emission kinetics and correlation with steady-state reaction (Alvarez-Pez, JM, Ballesteros, L., Talavera, E. & ^Yguerabide , J. According to fluorescence intensity (see J. Phys. Chem. A 105, 6320-6332 (2001)), it is suggested that the rate-determining step of pH-responsive behavior will be proton diffusion in the droplet. It is well known. The rate of proton diffusion in intracellular fluids is not significantly different from that in buffered solutions (Zaniboni, M. et al. Intracellular proton mobility and buffering power in cardiac ventricular myocytes from rat, rabbit, and guinea pig. Am. J. Considering Physiol. -Hear. means you can

생세포내 pH 모니터링을 위한 나노탐침의 적용 가능성Applicability of nanoprobes for pH monitoring in living cells

When the nanoprobe of the present invention was injected into living cells, the cells were not severely damaged even though the nanoprobe was deeply injected ( FIG. 7c ). For real-time observation of cell viability, HeLa cells were stained with calcein-AM and propidium iodide (PI), which emit green and red fluorescence in live and dead cells, respectively. Due to the micro-diameter (below 200 nm) and uniform structure (Fig. 1b), the nanoprobes of the present invention did not cause damage to cells during 10 min after insertion and extraction (Fig. 7c, upper panel). Importantly, since no red fluorescence signal was measured, the inventors found that the PI dye did not enter the intracellular space during nanowire insertion or even after extraction, confirming that the cell membrane was well preserved ( FIGS. 7C and 9 ). Moreover, the cell morphology was clearly not affected even after extraction, meaning that the pH detection system of the present invention did not induce membrane rupture and deformation.

Conversely, insertion of a typical cone-shaped tapered optical fiber (tip diameter: 200 nm or less) led to cell death due to membrane rupture with intracellular fluid leakage at the insertion point (Fig. 7c, lower panel and Fig. 9). When the cell viability was compared by inserting a nanoprobe and a tapered optical fiber into the cytoplasm and nucleus of HeLa cells, the nanoprobe of the present invention was a tapered optical fiber (33% in the cytoplasm (n=20), and 25% in the nucleus (n= 20)) showed a significantly higher cell viability (100% in the cytoplasm (n=20), 100% in the nucleus (n=20)) ( FIG. 10 ). The inventors confirmed that the cell viability upon insertion of the nanoprobe system was higher than that of existing systems used as carriers for bio-sensing probes (see Table 1).

Comparison of cell viability between the methods of the present invention and previously known methods used to provide various biosensing probes

유형category	세포 생존율cell viability	비고 note

나노탐침 nano probe	100%100%	본 발명the present invention
테이퍼진 광섬유 팁들Tapered Fiber Optic Tips	33%33%	본 발명the present invention
테이퍼진 광섬유 팁들Tapered Fiber Optic Tips	42%42%	Yan, R. etal.Nanowire-basedsingle-cellendoscopy.Nat.Nanotechnol.7, 191-196 (2011) 참조Yan, R. et al. Nanowire-based single-cellendoscopy. Nat. Nanotechnol. 7, 191-196 (2011)
bPEI (Branched polyethylenimine)Branched polyethylenimine (bPEI)	66%66%	Arif, M., Tripathi, S. K., Gupta, K. C. & Kumar, P. Self-assembled amphiphilic phosphopyridoxyl-polyethylenimine polymers exhibit high cell viability and gene transfection efficiency in vitro and in vivo. J.Mater.Chem.B1, 4020-4031 (2013) 참조Arif, M., Tripathi, SK, Gupta, KC & Kumar, P. Self-assembled amphiphilic phosphopyridoxyl-polyethylenimine polymers exhibit high cell viability and gene transfection efficiency in vitro and in vivo. See J.Mater.Chem.B 1, 4020-4031 (2013)
리포펙타민( Lipofectamine)Lipofectamine	36%36%
실리카 나노입자(Silica nanoparticle)Silica nanoparticles	90%90%	Wang, L. etal. A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells. Talanta209, 120436 (2020) 참조Wang, L. et al. A novel cell-penetrating Janus nanoprobe for ratiometric fluorescence detection of pH in living cells. See Talanta 209, 120436 (2020)
PS-co-PNIPAM 히드로겔(hydrogel)PS-co-PNIPAM hydrogel	85%85%	Liu, H. etal. Dual-emission hydrogel nanoparticles with linear and reversible luminescence-response to pH for intracellular fluorescent probes. Talanta 211, 120755 (2020) 참조Liu, H. et al. Dual-emission hydrogel nanoparticles with linear and reversible luminescence-response to pH for intracellular fluorescent probes. See Talanta 211, 120755 (2020)
GO 글리코시트(glycosheets)GO glycosheets	65%65%	Ji, D. K. etal. Targeted Intracellular Production of Reactive Oxygen Species by a 2D Molybdenum Disulfide Glycosheet. Adv. Mater .28, 9356-9363 (2016) 참조Ji, DK et al. Targeted Intracellular Production of Reactive Oxygen Species by a 2D Molybdenum Disulfide Glycosheet. Adv. Mater. 28, 9356-9363 (2016)

Next, the inventors verified that the pH monitoring through the nanoprobe guarantees high accuracy even in the complex cellular environment (Fig. 7d). To systematically manipulate intracellular pH, HeLa cells were treated with K ⁺ /H ⁺ -ionophore nigericin (Llopis, J., McCaffery, JM, Miyawaki, A., Farquhar, MG & Tsien, RY). Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA 95, 6803-6808 (1998)). Incubated in high potassium buffer solution. By measuring the fluorescence signal intensity ratio (I ₅₃₅ /I ₆₈₅ ) in HeLa cells at a specific pH value treated with nigericin in this way, the inventors successfully obtained a pH calibration curve for intracellular pH monitoring (black in Fig. 7d). curves and Fig. 11). From this curve, the inventors confirmed that the detectable range (pH 6.5-7.5) of the nanoprobe of the present invention is perfectly suitable for the detection and delivery of physiological pH in living cells (Jaworska, A., Malek, K. & Kudelski). , A. Intracellular pH - Advantages and pitfalls of surface-enhanced Raman scattering and fluorescence microscopy - A review. Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 251, 119410 (2021). Interestingly, in the nanoprobe of the present invention, almost the same PL intensity was measured in and outside living cells at the same pH 7.5 regardless of the surrounding environment (right inset of Fig. 7d and Fig. 12a,b,d); In the three experimental groups (pH 7, 7.5 and 8), the PL intensities obtained inside and outside the HeLa cells were also almost identical (Fig. 12 c, e). From this observation, the nanoprobe of the present invention can be used with various ions, proteins, and metabolites (Ellis, RJ Macromolecular crowding: An important but neglected aspect of the intracellular environment. Curr. Opin. Struct. Biol. 11, 114-119 (2001) ), suggesting that it can respond accurately to changes in pH even in complex cellular environments including

Fig. 8 shows the results of the light stability and reproducibility test of the nanoprobe. According to Fig. 8a, the PL peak intensity (I/I ₀ ), the change with time was hardly negligible. where I ₀ is the PL peak intensity at t=0. Figure 8b is the result of the measurement alternately in the acidic droplet (1x PBS, pH 5.0) and the basic droplet (1x PBS, pH 7.5). It was found that the reproducibility of the nanoprobe was excellent from the periodic fluctuation (n=3) of pH between 5.0 (white) and 7.5 (black).

9 is a comparison of cell viability between nanoprobe and tapered optical fiber insertion in living HeLa cells. As an indicator of living cells and dead cells, cells were treated with calcein-AM (green fluorescence) and propidium iodide (red fluorescence). was dyed with 9a to 9b show merged (brightfield and fluorescence) images upon insertion and extraction of a nanoprobe (a) or tapered optical fiber (b) (scale bar, 10 μm).

Figure 10 shows a histogram (n = 20) of cell viability after the inserted nanoprobe (grey) or tapered optical fiber (white) was extracted from the cytoplasm and nucleus, respectively. HeLa cells were propidium iodide as an indicator of dead cells. dyed with dyes. Cell viability is based on the number of viable cells without a red fluorescence signal.

Figure 11 shows a Boltzmann fitting for obtaining a pH calibration curve for the intracellular environment, Figure 11a is the Boltzmann equation of the pH calibration curve, Figure 11b is nigericin treated (nigericin) -treated) Boltzmann fitting curves (left graph) of the intensity ratio (I ₅₃₅ /I ₆₈₅ ) as a function of pH value of HeLa cells, and the value of each parameter (right table).

12 shows the results of investigation of pH-dependent fluorescence signals of nanoprobes outside and inside living HeLa cells. FIGS. 12A to 12B are bright fields of nanowire insertion sites outside (a) and inside (b) HeLa cells, respectively. Images (fixed pH: 7.5) are shown, and FIGS. 12c to 12e are PLs of nanoprobes measured outside (black line) and inside (grey line) of nigericin-treated HeLa cells at various pHs (7.0-8.0). Spectra are shown (scale bar, 10 μm).

13 shows the results of monitoring the pH values in the cytoplasm and the cell nucleus during the entire cycle of a single cell using the nanoprobe. According to FIG. 13a, the nanoprobe is the cytoplasm ( Inserted into the top) and nucleus (bottom) (scale bar 10 μm). 13B is a comparison diagram between cytoplasmic pH (n=15) and nuclear pH (n=29). 13c shows the identification process of cell cycle phases for individual HeLa cells, when Hoechst dye specifically stains the nucleus of live cells (step 1), using the automatic image segmentation algorithm of the present invention. Calculate the net fluorescence intensity of the nucleus for all cells (step 2), prepare a DNA histogram to profile the cell cycle of HeLa cells (step 3), and color mapping on the cell image for each cell was identified (step 4) (scale bar 50 μm). 13D shows the measured nuclear pH for each cell cycle stage. Schematic (top) of cell cycle stages, dark field and merged (bright field + fluorescence) images of Hoechst stained cells (middle), and nuclear pH values (bottom) are shown for cell cycle stages. G1 and S/G2 phases showed similar pH values (G1 phase: 6.91±0.03 (n=14), S/G2 phase: 6.92±0.03 (n=15)), prophase, metaphase, Nuclear pH values in telophase, and cytoplasmic division, showed tidal curves (early: 6.97±0.05 (n=10); middle: 7.01±0.05 (n=10); late: 7.05±0.03). (n=12); cytoplasmic division: 6.98±0.03 (n=16)) (scale bar 10 μm).

Since the nanoprobe of the present invention can monitor the local pH of different organelles in real time, the inventors were able to successfully demonstrate the measurement of pH values for the cytoplasm and nucleus in a single living cell ( FIGS. 13a to 13b ). Critical cellular functions (e.g., DNA replication, gene expression, and epigenetic modulation (Francastel, C., Schubeler, D., Martin, DIK & Groudine, M. Nuclear compartmentalization and gene activity. Nat. Rev. Mol ) Cell Biol. 1, 137-143 (2000); and Nakamura, A. & Tsukiji, S. Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein. Bioorganic Med. Chem. Lett. 27, 3127-3130 (2017)), direct determination of nuclear intracellular pH has been extremely difficult (Casey, JR, Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular pH. Nat. Rev. Mol. Cell Biol. 11, 50-61 (2010)), the main reason is that two membranes, the cell membrane and the nuclear membrane, exist in the cell. For this reason, many studies have assumed that the nuclear pH is equal to the cytoplasmic pH (Casey, JR, Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular pH. Nat. Rev. Mol. Cell Biol. 11, 50 -61 (2010); and Fabre, E. & Hurt, EC Nfuclear transport . Current Opinion in Cell Biology vol. 6 (EMBL, Heidelberg, 1994). The ability to control the internal pH of the nuclear compartment It has been suggested that pH can be made different from cytoplasmic pH (Sherman, TA, Rongali, SC, Matthews, TA, Pfeiffer, J. & Nehrke, K. Identification of a nuclear carbonic anhydrase in Caenorhabditis elegans. Biochim. Biophys. Acta - Mol. Cell Res. 1823, 808-817 (2012); Santos, JM, Martinez-Zaguilαn, R., Facanha, AR, Hussain, F. & Sennoune, SR Vacuolar H+-ATPase in the nuclear membranes regulates nucleo-cytosolic proton gradients. Am. J. Physiol. - Cell Physiol. 311, C547-C558 (2016); and Nakamura, A. & Tsukiji, S. Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein. Bioorganic Med. Chem. Lett. 27, 3127-3130 (2017)). To answer this controversial question, the inventors inserted a nanoprobe at a desired location in a single live HeLa cell and measured the pH values of the nucleus and cytoplasm separately (Fig. 13a). As a result, the inventors found that the nuclear pH (6.92 ± 0.04, n = 29) was significantly lower than the cytoplasmic pH (7.11 ± 0.05, n = 15) (Fig. 13b), which contributed to the separate pH control function for each cell compartment. This means that there may be a pH gradient between the nucleus and the cytoplasm.

Because the nuclear membrane was easily pierced by the nanoprobe without leakage despite its robustness, the inventors were able to directly monitor changes in nuclear pH across the entire human cell cycle. For this, in advance, it was necessary to confirm the cell cycle status of individual HeLa cells ( FIGS. 13c to 13d ) (Rloukos, V., Pegoraro, G., Voss, TC & Misteli, T. Cell cycle staging of individual cells). by fluorescence microscopy (see Nat. Protoc. 10, 334-348 (2015)). In principle, the total amount of DNA inside the nucleus changes as cell division progresses, and quantifying the total amount of DNA can determine the cell cycle phase of cell division. Specifically, the inventors first stained the cells with Hoechst dye that specifically binds to DNA inside the nucleus and emits a blue fluorescent signal (step 1), and automatic image analysis (nuclei segmentation) was performed. The DNA content of each cell was measured through the assay, where the total fluorescence intensity was calculated for several nuclei (see Figure 13c and Methods). Finally, cell cycle stages were identified by color mapping to cell images based on DNA histograms (step 4). Through the analysis, we identified the cell cycle phases (G1, S and G2/M) of individual HeLa cells, and then calculated the proportions of each phase (G1, 73.9%, S, 11.1%, G2/M, 15.0%). and the ratio of each step was in good agreement with known HeLa cell properties (G1, 72.1%; S, 12.6%; G2/M, 12%) (Athukorala, Y., Trang, S., Kwok, C. & Yuan, YV Antiproliferative and antioxidant activities and mycosporine-Like amino acid profiles of wild-Harvested and cultivated edible canadian marine red macroalgae. Molecules 21, (2016)).

Based on the evaluation of each cell cycle stage (Fig. 13c), the inventors measured nuclear pH changes during cell division to find resting pH homeostasis and mitotic pH fluctuations (Fig. 13d). Specifically, HeLa cells in the G1 and S/G2 stages showed similar pH values (G1 stage: 6.91±0.03 (n=14); S/G2 stage: 6.92±0.03 (n=15), bottom of FIG. 13d ) gray box). Previously, several studies have reported that cytoplasm during resting phase exhibits pH fluctuations for several reasons such as ATP synthesis/hydrolysis and redox oscillations (Da Veiga Moreira, J. et al. Cell cycle progression is regulated by intertwined redox oscillators). Theor. Biol. Med. Model. 12, 1-14 (2015); and DeBerardinis, RJ, Lum, JJ, Hatzivassiliou, G. & Thompson, CB The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation.Cell Metab 7, 11-20 (2008)) . Unlike before, we clearly observed that the nucleus preserves pH without pH fluctuations in the G1 and S/G2 phases, probably due to the pH-regulating function of the nuclear membrane. This quiescent nuclear pH homeostasis is a result of previous findings investigating changes in nuclear pH in budding yeast (Zhao, H. et al. Dynamic imaging of cellular pH and redox homeostasis with a genetically encoded dual-functional biosensor, pHaROS, in yeast). J. Biol. Chem. 294, 15768-15780 (2019)). Surprisingly, when HeLa cells entered prophase, the nuclear pH continued to increase slightly until the cells reached telophase (previous: 6.97±0.05 (n=10), metaphase: 7.01±0.05 ( n=10), late: 7.05±0.03 (n=12), bottom white box in FIG. 13D , and FIGS. 14-15 ). During mitosis, transient disruption of nuclear pH homeostasis may be associated with disruption of the nuclear membrane (see Cooper GM. The Cell: A Molecular Approach. 2nd edition. (Sunderland (MA), Sinauer Associates, 2000)), temporarily It can interfere with the ability of the nucleus to regulate pH. However, as the cells reached the mitotic stage at the end of mitosis, the nuclear pH returned to the original pH value (cytoplasmic: 6.98 ± 0.03 (n = 16)), indicating that the reorganization of the nuclear membrane of the divided cell was originally suggest that it will lead to the restoration of pH homeostasis. By directly monitoring the pH during the entire cell cycle, the inventors clearly confirmed that the nucleus has its own pH control function.

14 shows brightfield images (top panel) and merged (brightfield and fluorescence, bottom panels) images of insertion of a nanoprobe into a single HeLa living cell during mitosis, observed with confocal microscopy (scale bar, 10 μm). ).

Figure 15 shows merged (bright field and fluorescence) images observed with confocal microscopy of HeLa cells stained with a nuclear-specific Hoechst dye (white) during mitosis (from electrophoresis to cytoplasmic division) for pH measurement (Fig. Scale bar, 10 μm).

FIG. 16 shows changes in cytoplasmic pH in response to external calcium ions, and shows real-time cellular pH monitoring results for ion stress. 16A is a schematic diagram of intracellular acidification in the presence of excess calcium ions. In general, high concentrations of calcium ions affect pH homeostasis by adversely affecting cells, including overproduction of adenosine triphosphates (ATPs) and reactive oxygen species (ROS). 16b to 16c, different cellular responses to external calcium ion stress were measured by changes in cytoplasmic pH (n=3). The white triangle in the bright field image indicates the position where the nanoprobe of the present invention is inserted for pH measurement. Gray and black arrows indicate the point of introduction and removal of external Ca ²⁺ stress, respectively (scale bar 10 μm).

In addition, as a result of examining the cytoplasmic pH dynamics of single HeLa living cells by providing external divalent ion stress, the inventors confirmed that individual cells actually react differently depending on the ion ( FIG. 16 ). When an excess of calcium (5 mM) was added to the cell culture medium, the cytoplasmic pH significantly decreased within 30 min as a result of intracellular acidification induced by high extracellular Ca ²⁺ (7.17±0.02 - 6.97±0.04). (Fig. 16b). Interestingly, when Ca ²⁺ was replaced with Mg ²⁺ , the pH change was insignificant (7.09±0.01 - 7.08±0.01) ( FIG. 17 ). It is known that the presence of excess Ca ²⁺ in the extracellular medium can lead to the production of reactive oxygen species (ROS), mitochondrial dysfunction by increasing ATP levels, and even apoptosis through apoptosis and necrosis. McGinnis, KM, Wang, KKW & Gnegy, ME Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells. J. Neurochem. 72, 1853-1863 (1999); and Voccoli, V. , Tonazzini, I., Signore, G., Caleo, M. & Cecchini, M. Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death. See Cell Death Dis. 5, 1-10 (2014)) . Therefore, Ca ²⁺ -dependent intracellular acidification of HeLa cells was considered to be caused by the side effect of high extracellular Ca ²⁺ , which was further supported by the investigation of cell viability following calcium ion treatment (Fig. 18a). Moreover, the magnesium treatment experiment of the present invention showed that cells are resistant to an increase in extracellular Mg ²⁺ concentration, and the previous results (Libako, P. et al. Blocking the rise of intracellular calcium inhibits the growth of cells cultured in Consistent with different concentrations of magnesium. Magnes. Res. 25, 12-20 (2012)), unlike Ca ²⁺ stimulation, there was no cell malfunction or apoptosis ( FIGS. 17 and 18b ).

Importantly, living HeLa cells restored their original pH state when the external ionic stress was removed (Fig. 16c). To observe the restoration of pH homeostasis, the inventors incubated HeLa cells with an excess of Ca ²⁺ (5 mM) for 30 min, and then quickly adjusted the Ca ²⁺ concentration of the medium to the normal range (1.8 mM). During this process, the inventors monitored changes in cytoplasmic pH in three individual cells. As observed in the previous Ca ²⁺ -dependent intracellular acidification ( FIG. 16b ), high extracellular Ca ²⁺ during the first 30 min induced the cytoplasm of HeLa cells to be acidic (7.10±0.02 - 6.99±0.02). Remarkably, after removal of extracellular Ca ²⁺ stress (Fig. 16c, black arrow), cells gradually restored their intrinsic neutral pH (6.99±0.02 - 7.09±0.02), indicating that the cytoplasmic pH homeostasis of living HeLa cells had previously been It means a successful recovery from the loss of pH control caused by ionic stress. Although the overall tendency of HeLa cells to external ionic stress was similar, it is interesting to note that individual responses of HeLa cells, expressed as pH, were heterogeneous, such as size, morphology, and intercellular differences such as neighboring cells and division phase (Kultz, D. Molecular and evolutionary basis of the cellular stress response (see Annu. Rev. Physiol. 67, 225-257 (2005)).

17 shows the results of real-time measurement of the cytoplasmic pH of HeLa cells treated with an excess of magnesium ions (5 mM). A bright field image (upper panel) obtained by tracking a single HeLa cell (n=3) and the measured pH It shows the change (lower graph). The white triangle in the upper panel shows the insertion position of the nanoprobe. Gray arrows indicate the point at which the medium is switched from a normal magnesium concentration (1.8 mM) to a high magnesium concentration (5 mM) (scale bar, 10 μm).

18a to 18b show merged (bright-field and fluorescence) and dark-field images of live HeLa cells treated with excess calcium ions (a) and excess magnesium ions (b), both assays showing HeLa cells Cell viability was analyzed by transstaining with Sein-AM and propidium iodide (scale bar, 100 μm). From this, it was confirmed that although calcium ion treatment puts stress on HeLa cells and reduces the survival rate, magnesium ion treatment does not affect the survival rate of HeLa cells.

결론conclusion

Utilizing the nanoprobe with local pH detection and delivery in the present invention, the inventors were able to monitor pH changes by successfully accessing organelles and cytoplasm without causing cell damage and leakage in single living cells. Beyond impermeable cell and nuclear membranes, the in situ pH monitoring of the present invention is important in that it can provide a fundamental understanding of the role of intracellular organelle membranes. As a result of observing the pH difference between the cytoplasm (7.11±0.05) and the nucleus (6.92±0.04), it was confirmed that the cell activity can exhibit different pH changes by the nuclear membrane (Sherman, TA, Rongali, SC, Matthews, TA). , Pfeiffer, J. & Nehrke, K. Identification of a nuclear carbonic anhydrase in Caenorhabditis elegans. Biochim. Biophys. Acta - Mol. Cell Res. 1823, 808-817 (2012); Santos, JM, Martinez-Zaguilαn, R. , Facanha, AR, Hussain, F. & Sennoune, SR Vacuolar H+-ATPase in the nuclear membranes regulates nucleo-cytosolic proton gradients. Am. J. Physiol.-Cell Physiol. 311, C547-C558 (2016); and Nakamura, A. & Tsukiji, S. Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein. Bioorganic Med. Chem. Lett. 27, 3127-3130 (2017)). In particular, pH homeostasis and fluctuations in the process of cell growth and nuclear division promote biomolecular synthesis as the nuclear membrane before decomposition is involved in intracellular pH maintenance and nuclear transport (Cooper GM. The Cell: A Molecular Approach. 2nd edition. (Cooper GM. The Cell: A Molecular Approach. 2nd edition.) Sunderland (MA), Sinauer Associates, 2000); and Demaurex, N. pH homeostasis of cellular organelles. News Physiol. Sci. 17, 1-5 (2002)). To the best of the inventors' knowledge, this is the first direct evidence of the existence of an independent pH control function, especially in the dividing nucleus of human cells.

As observed in different cellular responses to external ion stimuli, the local pH monitoring nanoprobe of the present invention can be widely applied to study the life of individual cells in a variety of interesting conditions. For example, real-time detection of changes in the pH of organelles during various cell behaviors (e.g., differentiation, cell signaling or communication, programmed cell death) could be utilized to understand biological processes along organelle membranes (Jaworska, et al. A., Malek, K. & Kudelski, A. Intracellular pH - Advantages and pitfalls of surface-enhanced Raman scattering and fluorescence microscopy - A review. Spectrochim. Acta - Part A Mol. Biomol. Spectrosc. 251, 119410 (2021); and Han, J. & Burgess, K. Fluorescent indicators for intracellular pH. Chem. Rev. 110, 2709-2728 (2010)).

방법Way

시약 및 재료:Reagents and Materials:

Poly(vinylbenzyl chloride) (PVC, M _n = 55,000 g/mol), sodium azide, N,N-dimethylformamide (DMF), 1-methyl-2-pyrrolidinone (NMP) , methanol, dimethyl sulfoxide-d ₆ (DMSO-d ₆ ), agar powder, 10X phosphate buffered saline (PBS), sodium hydroxide, hydrochloric acid (37%), propidium iodide, calcein-AM, and nigericin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO). 5'-DBCO-T ₅ -FAM-3' was synthesized by Bioneer (Daejeon, Korea). HEPES (pH 7.5) buffer (1 M), potassium chloride, calcium dichloride (CaCl ₂ ) and magnesium dichloride (MgCl ₂ ) were purchased from BioPrince (Chuncheon, Korea). Hoechst 33342 (10 mg/ml) solution was purchased from Biotium (Fremont, CA). Glass capillaries (BF-100-50-10) for nanopipette fabrication were purchased from Sutter Instrument (Novato, CA).

나노탐침의 제작:Fabrication of the nanoprobe:

A mixture of PVC (0.014 g, 131 mmol) and sodium azide (0.010 g, 220 mmol) in anhydrous DMF solvent (0.7 mL) was mixed in an amber vial at 70 °C and covered with aluminum foil to block light. After 2 hours of reaction, methanol (0.5 mL) was added, and the mixed solution was centrifuged at 10,000 rpm for 1 minute (Mini microcentrifuge, Labogene) to remove excess unreacted reagent and azide-functionalized polymer. was precipitated. Finally, the obtained precipitate was dried under vacuum for 1 hour and then dissolved by adding NMP solvent (50 μL). Successful synthesis of PVBN ₃ was confirmed by ¹ H NMR spectroscopy ( FIG. 2 ). A glass nanopipette was processed using a P-97 micropipette puller (Sutter Instrument) for nanowire fabrication, and a tapered optical fiber was manufactured using a P-2000 laser-based micropipette puller (Sutter Instrument). Then, the position of the glass nanopipette and the tapered optical fiber was precisely controlled by an xyz stepping motor stage with a position accuracy of sub-250 nm (Kohzu Precision). For nanowire fabrication, a glass nanopipette filled with NMP's PVBN ₃ solution at a concentration of 1.0 wt% was vertically lowered to touch the tip of the tapered optical fiber. When the nanopipette was lifted in the vertical direction, PVBN ₃ nanowires were formed at the tip of the tapered optical fiber by rapid solvent evaporation, thereby forming freestanding PVBN ₃ nanowires. Nanowire fabrication is self-optical imaging consisting of a two-axis CCD camera (INFINITY 1-2C, Lumenera Camera), an objective (100x Plan Apo infinitely calibrated objective, Mitutoyo), and a yellow LED illuminator (precision LED spotlight, 590 nm, Mightex). was monitored in real time using the system.

나노선에 대한 플루오레세인의 접합:Conjugation of Fluorescein to Nanowires:

To bond DBCO fluorescein (FAM) to PVBN ₃ nanoprobes, a glass micropipette was filled with an aqueous solution (100 nM) containing DBCO-FAM molecules. When the glass micropipette was vertically lowered and the nanowire was immersed for 10 minutes, the DBCO-FAM molecule was bonded to the azide group of the PVBN ₃ nanowire by a click reaction. By adjusting the contact area between the nanowire and the solution containing DBCO-FAM molecules, the FAM-labeled area of the nanoprobe could be controlled. Nanoprobes were washed twice with 1x PBS solution prior to pH measurement analysis.

나노탐침을 통한 형광 신호(PL 스펙트럼) 측정:Measurement of fluorescence signal (PL spectrum) with nanoprobe:

A continuous laser (473 nm blue solid-state laser, MBL-III-473, Uniotech) coupled with a computer-controlled shutter to excite the DBCO fluorescein signal (PL spectrum) from the tip of the nanoprobe was coupled to a fiber optic and a 1x2 optical coupler (narrowband fiber optic). coupler, 532±15 nm, 50:50 split, Thorlab) was injected into the nanoprobe. All PL spectra were recorded with a spectrometer (Avaspec-ULS2048L-EVO, Avantes).

세포 배양 실험:Cell culture experiments:

HeLa cells were obtained from the Korean cell line bank. Cells were cultured in 35 mm Petri dishes (SPL Life Sciences) under appropriate conditions (37° C. temperature and 5% CO ₂ atmosphere), 10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin (Welgene), and 100 μg/ml strepto Cultured in Dulbecco's modified Eagle's medium (DMEM, Welgene) supplemented with mycin (Welgene). In preparation for cell experiments, HeLa cells were cultured for two days.

세포 생존율 분석:Cell viability assay:

To analyze cell viability, HeLa cells were pre-incubated with calcein-AM and propidium iodide dyes at 37°C for 15 min. To investigate the insertion effect of nanoprobes and tapered optical fibers on HeLa cells, both were inserted into the cytoplasm or nucleus of HeLa cells for 1 min and then extracted. After this process, green fluorescence (515 nm) and red fluorescence (636 nm) were observed with a confocal microscope (STELLARIS 5, Leica) using a 10x objective lens (0.4 numerical aperture, HC PL APO 10x, Leica) to determine the cell viability. evaluated. In cell viability histogram investigation, cells were cultured in cell culture conditions for 3 hours and then imaged by confocal microscopy.

검정 곡선을 얻기 위한 세포 내 pH 조작:Intracellular pH manipulation to obtain a calibration curve:

Cultured HeLa cells were washed twice with freshly prepared DMEM and nigericin buffer (10 mM HEPES, 10 mM NaCl, 130 mM KCl, 1 mM MgCl ₂ ) at various pH values (5-9). Next, we added 15 μM nigericin to the washed cells at 37° C. for 15-25 minutes. Based on the sigmoidal increase in the fluorescence signal intensity ratio dependent on the different pH-dependent fluorescence signals (5-9), the pH calibration curve is calculated from the measured data (R ² = 0.9969) calculated by the IUPAC definition. ), very good sensitivity (18.722 (I ₅₃₅ /I ₆₈₅ )/pH unit), and a good correlation with detection resolution (0.0365 pH unit) was obtained by Boltzmann fitting.

나노탐침을 단일 헬라 세포에 삽입하기 위한 구성:The configuration for inserting the nanoprobe into a single HeLa cell:

Before cell experiments, cultured HeLa cells were washed twice with freshly prepared DMEM. To precisely control the insertion site of the nanoprobe inside a single HeLa cell, a self-microscopic photoluminescence configuration consisting of an x-y-z micromanipulator (positional accuracy: 250 nm, Kohzu Precision), a motor controller (SC-210, Kohzu Precision) and a computer. was used to accurately position the nanoprobe. During insertion, the position of the nanoprobe was monitored with a 10x objective (0.4 numerical aperture, HC PL APO 10x, Leica) and a confocal microscope (STELLARIS 5, Leica) equipped with a CCD camera. PL spectra were collected in real time while positioning the nanoprobe at a desired position inside the cell.

개별 헬라 세포의 세포 주기 상태 식별:Identify the cell cycle status of individual HeLa cells:

To specifically stain DNA in HeLa cells, first a diluted Hoechst 33342 solution (10 μg/ml) was prepared and then mixed with the cultured cells for 15 minutes (in cell culture conditions). Images of stained cells were acquired with a confocal microscope at 2048 x 2048 pixels. The nuclear fluorescence intensity of each cell was calculated by applying a MATLAB-based image processing algorithm for nuclear division. Specifically, the algorithm is designed to binarize the filtered image by removing noise from the raw image using Gaussian filtering and setting an adaptive threshold. The binary image was then segmented by applying an open and close algorithm to smooth out the rough edges. To minimize the identification error of individual nuclear segmentation, small binary noise clusters and nuclei around the boundary region of the image process were automatically removed. Based on the automatically segmented images, the fluorescence intensity within segmented regions of each nucleus was collected. From the fluorescence intensity data, individual cells were visually selected cutoff (Roukos, V., Pegoraro, G., Voss, TC & Misteli, T. Cell cycle staging of individual cells by fluorescence microscopy. Nat. Protoc. 10, 334-348). (2015)) sorted DNA histograms into different cell cycle stages (G1, S, G2/M) were plotted. Here, the proportion of cells within each stage was automatically calculated using the Origin software (version 8.5). The phase of each cell in the image was divided into G phase, S phase, and G2/M phase by color mapping to cell images of different colors based on a DNA histogram.

세포 주기 동안 핵 pH 변화의 측정:Measurement of changes in nuclear pH during the cell cycle:

The cultured HeLa cells were washed twice with freshly prepared DMEM and incubated with Hoechst dye-containing buffer (10 μg/ml in DMEM) for 15 min. After the medium was replaced with fresh DMEM buffer, the nanoprobes were inserted into single HeLa viable cells at each cell cycle stage and the nuclear pH was measured by imaging with a confocal microscope.

Although an embodiment of the present invention has been described above, the contents described so far are merely illustrative of some of the preferred embodiments of the present invention, and are described above except as may appear in the claims appended below. It is not limited by one content. Accordingly, the present invention understands that many changes, modifications and substitutions of equivalents can be made within the scope set forth in the following claims by those skilled in the art without departing from the spirit and spirit of the invention. will have to

부호의 설명Explanation of symbols

1: nano probe

2: optical fiber

2a: first optical fiber

2b: second optical fiber

3: tapered tip

4: light source

5: optical coupler (fiber coupler)

6: manipulator

7: single living cell

8: spectrometer

Claims

A method for manufacturing a nanoprobe comprising:

(a) filling a nanopipette with a nanowire material solution and lowering the nanopipette to bring the nanowire material solution into contact with an end of an optical fiber;

(b) raising the nanopipette to grow a nanowire on the end of the optical fiber;

(c) filling a micropipette with an aqueous solution containing a pH-responsive fluorescent substance and lowering the micropipette so that a part of the nanowire is immersed in the aqueous solution; and

(d) raising the micropipette to form a nanoprobe labeled with a pH-responsive fluorescent material.
The method of claim 1,

The nanowire material solution is a hydrophobic polymer solution, a method of manufacturing a nanoprobe.
3. The method of claim 2,

The hydrophobic polymer solution is at least PVBN 3 , PVB-alkyne, PVB-COOH is selected from the group consisting of, nanoprobe manufacturing method.
The method of claim 1,

The optical fiber has a tapered tip, nanoprobe manufacturing method.
The method of claim 1,

The pH-responsive fluorescent material is a fluorescein molecule having a functional group capable of binding to the nanowire.
6. The method of claim 5,

Wherein the fluorescein is selected from the group consisting of at least DBCO-FAM, Azide-FAM, and Amine-FAM.
The method of claim 1,

Wetting (or labeled) length of the nanowire by the pH-responsive fluorescent material is controlled to 100 to 900nm, nanoprobe manufacturing method.
The method of claim 1,

Wetting (or labeled) length of the nanowire by the pH-responsive fluorescent material is controlled to 100 to 500nm, nanoprobe manufacturing method.
A nanoprobe for pH measurement, comprising:

optical fiber;

a nanowire formed by growing a nanowire material solution at one end of the optical fiber; and

A nanoprobe comprising a pH-responsive fluorescent material labeled on a part of the nanowire.
10. The method of claim 9,

The nanowire material solution is a hydrophobic polymer solution, nanoprobe.
11. The method of claim 10,

The hydrophobic polymer solution is at least PVBN 3 , PVB-alkyne, PVB-COOH, nanoprobes selected from the group consisting of.
10. The method of claim 9,

One end of the optical fiber has a tapered tip, a nanoprobe.
10. The method of claim 9,

The pH-responsive fluorescent material is a fluorescein molecule having a functional group capable of binding to the nanowire, nanoprobe.
14. The method of claim 13,

The fluorescein is at least selected from the group consisting of DBCO-FAM, Azide-FAM, Amine-FAM, nanoprobe.
10. The method of claim 9,

The wetting (or labeled) length of the nanowire by the pH-responsive fluorescent material is controlled to be 100 to 900 nm, the nanoprobe.
10. The method of claim 9,

The wetting (or labeled) length of the nanowire by the pH-responsive fluorescent material is controlled to be 100 to 500 nm, the nanoprobe.
10. The method of claim 9,

The nanoprobe is characterized in that the diameter is uniform, the nanoprobe.
10. The method of claim 9,

The nanoprobe is characterized in that the diameter of 10nm to 900nm, nanoprobe.
10. The method of claim 9,

The nanoprobe is characterized in that the diameter of 10nm to 400nm, the nanoprobe.
10. The method of claim 9,

The nanoprobe is characterized in that the length of 1μm to 10μm, nanoprobe.
10. The method of claim 9,

The nanoprobe is characterized in that the length of 1μm to 5μm, nanoprobe.
A method for measuring pH in a single cell, comprising:

(a) inserting a nanoprobe formed by combining a pH-responsive fluorescent material capable of responding to pH to a nanowire grown on a tapered tip of an optical fiber into a single cell;

(b) incident light to the nanoprobe through the optical fiber;

(c) excitation of the pH-responsive fluorescent material by the light to generate fluorescence;

(d) acquiring a fluorescent signal generated from the fluorescent material through the optical fiber according to the pH of the cell; and

(e) analyzing the fluorescence signal to obtain an intracellular pH value, a method for measuring pH in a single cell.
23. The method of claim 22,

A method for measuring pH in a single cell, characterized in that the fluorescence signal acquired through the optical fiber is transmitted to a spectrometer via an optical coupler.
23. The method of claim 22,

The method for measuring pH in a single cell, characterized in that the measurement of the pH value is obtained from spectral data of fluorescence in the spectrometer.
23. The method of claim 22,

Light incident through the optical fiber is light in the near-infrared or visible region, characterized in that the pH measuring method in a single cell.
23. The method of claim 22,

Light incident through the optical fiber is characterized in that the wavelength of 300nm to 1000nm, pH measuring method in a single cell.
23. The method of claim 22,

Light incident through the optical fiber is characterized in that the wavelength of 400nm to 700nm, pH measuring method in a single cell.
A device for measuring pH in a single cell, comprising:

a nanoprobe formed by combining a pH-responsive fluorescent material capable of responding to pH to a nanowire grown on the tapered tip of an optical fiber;

a manipulator capable of three-dimensional movement control of the nanoprobe to insert the nanoprobe into the single living cell;

a light source for applying light to the optical fiber;

an optical coupler connecting the optical fibers to transmit the light incident through the optical fiber to the nanoprobe and to transmit the fluorescence signal generated from the nanoprobe to the spectrometer through an additional optical fiber; and

A single intracellular pH measuring device comprising a spectrometer for obtaining a pH value by analyzing spectral data from the fluorescence signal generated by the nanoprobe.
A method for preparing a nanowire material solution according to any one of claims 1 to 3, comprising:

Mix a mixture of PVC (0.014 g, 131 mmol) and sodium azide (0.010 g, 220 mmol) in anhydrous DMF solvent (0.7 mL) in an amber vial at 70 °C and cover with aluminum foil to block light. step;

After 2 hours of reaction, methanol (0.5 mL) is added, and the mixed solution is centrifuged at 10,000 rpm for 1 minute to remove excess unreacted reagent and to precipitate an azide-functionalized polymer; and

A method comprising the step of drying the obtained precipitate under vacuum for 1 hour and then dissolving it by adding NMP solvent (50 μL).