Method for measuring multiple physical fields in living cells by controlling single nano-diamond

Abstract

The invention discloses a method for measuring multiple physical fields in living cells by controlling a single nano-diamond, which comprises the following steps: capturing the nano-diamond through a nano-electrode to form a probe, and controlling the probe to move the nano-diamond into a living cell sample; measurement of multiple physical fields in living cells is realized by measuring the spinning property of the nano-diamond at the tip of the probe. According to the method, active regulation and control of the spatial position of the nano-diamond in the living cell can be realized, the spatial number distribution of the nano-diamond is optimized so as to improve the measurement resolution, accurate calibration of the nano-diamond is ensured, long-time measurement of any position in the cell is allowed, and meanwhile, the cell activity is maintained, so that the cell can normally react to external stimulation.

Description

Technical Field

[0001] The present invention belongs to the technical field of quantum sensing, and particularly relates to a method for manipulating single nanodiamonds to measure multi-physical fields in living cells. Background Art

[0002] Nanodiamonds are diamond particles with nanoscale particle sizes. Like larger-sized diamonds, they are all composed of Sp3 hybridized carbon atoms, so they have stable chemical properties. Based on this, it has been found that nanodiamonds have good biocompatibility, are not prone to reactions in living cells that may cause potential toxic effects, and are not easily decomposed, thus can stably exist in living cells. Utilizing this property, nanodiamonds are widely used in intracellular labeling or drug delivery, providing important value in the research of living cells.

[0003] In addition to the above applications, nanodiamonds have also been used as a kind of sensor in the research of living cells in recent years. This is by using a special spin color center system in nanodiamonds. This type of color center has the effect of optically detected magnetic resonance, making its fluorescence properties affected by physical quantities such as temperature, magnetic field, electric field, and pressure in the environment. By measuring its fluorescence properties, the changes of relevant physical quantities in the environment can be deduced. Due to the nanoscale size of nanodiamonds and the atomic-scale size of spin color centers, the measurement using nanodiamonds containing spin color centers can achieve extremely high spatial resolution. In addition, in systems such as living cells and living organisms, there is often a high autofluorescence background, while the fluorescence of spin color centers is stable and bright, which can resist the interference of the autofluorescence background and achieve accurate fluorescence measurement. In the earliest research, a nanothermometry method in living cells based on nitrogen-vacancy defect color centers was proposed, and the feasibility of nanodiamonds as sensors in cells was verified. Subsequently, a series of methods such as in-situ temperature-controlled photothermal therapy-induced embryo division, in-situ monitoring of mitochondria in living cells, and intracellular thermal conductivity measurement based on nanodiamonds containing spin color centers have been successively proposed, fully demonstrating the application prospects of nanodiamonds containing spin defect color centers as sensors in living cells.

[0004] Although the technology has become increasingly mature after more than a decade of development, the measurement of nanodiamonds containing spin color centers in living cells or in vivo is still subject to some limitations. One very important point is the manipulation of the spatial position of nanodiamonds in living cells. Currently, the measurement of nanodiamonds containing spin color centers in living cells lacks an active regulation scheme for the spatial position of nanodiamonds. Existing technologies usually use microinjection or endocytosis to introduce nanodiamonds containing spin color centers into the interior of living cells, and then track and measure the nanodiamonds containing spin color centers that freely drift within the cells. Since such technologies cannot regulate the spatial position of nanodiamonds containing spin color centers, they cannot achieve continuous observation of specific regions, nor can they easily achieve instant measurement of regions of interest. Methods of surface modification have been reported to enable nanodiamonds containing spin color centers to be specifically labeled on specific organelles. However, the modification process of this method is complex, and the number distribution of nanodiamonds on the organelles cannot be controlled, easily leading to uneven spatial distribution of nanodiamonds containing spin color centers, thereby reducing the spatial resolution of the measurement, and there will be "measurement dead zones". In addition, although nanodiamonds containing spin color centers have a small size, their structure is dense and has a comparable mass to organelles usually composed of membrane structures, which may have a potential impact on the physiological processes of organelles.

[0005] In addition, existing methods capable of actively regulating the spatial position of nanodiamonds mainly include optical tweezers, magnetic tweezers, anti-Brownian electrokinetic traps, atomic force microscope probes, etc. However, due to configuration or experimental condition limitations, these methods cannot be applied to regulate the spatial position of nanodiamonds in living cells. Therefore, it is necessary to explore methods that can actively regulate the spatial position of nanodiamonds in living cells to enable measurement in living cells. Summary of the Invention

[0006] In view of the above, the purpose of the present invention is to provide a method for manipulating single nanodiamonds to measure multiple physical fields in living cells, realizing active regulation of the spatial position of nanodiamonds in living cells, optimizing their spatial number distribution to improve measurement resolution, ensuring accurate calibration of nanodiamonds, allowing long-term measurement of any position within the cell, while maintaining cell viability so that it can respond normally to external stimuli, providing new tools and means for biomedical research and other directions.

[0007] To achieve the above invention purpose, the technical solution provided by the present invention is as follows:

[0008] A method for manipulating single nanodiamonds to measure multiple physical fields in living cells provided by an embodiment of the present invention includes the following steps:

[0009] Capture a nanodiamond through a nanoelectrode to form a probe, and manipulate the probe to move the nanodiamond into a living cell sample;

[0010] By measuring the spin properties of the nanodiamond at the tip of the probe, the measurement of multi-physical fields inside living cells is achieved.

[0011] Preferably, the nanoelectrode includes a plurality of independent electrodes with roots fixed on an insulating housing, and the nanodiamond is captured and manipulated through the plurality of independent electrodes.

[0012] Preferably, the insulating housing includes a plurality of cavities complementary in shape to the independent electrodes, enabling the roots of the independent electrodes to be installed in the cavities. The size of the longest dimension of the insulating housing is slightly smaller than the size of the longest dimension of the independent electrodes, exposing the tips of the independent electrodes and making the tips of the plurality of independent electrodes lie in the same plane and the connecting lines of the tip centers form a regular polygon, and the size of the regular polygon is comparable to the size of the nanodiamond. The resistance of the insulating housing is greater than or equal to 10 10 Ωm, and the material used is non-cytotoxic and can stably exist inside cells.

[0013] Preferably, the cross-section of each independent electrode gradually decreases from the root to the tip, and the tip is a sphere with a radius of curvature in the range of 1 - 1000 nm. The material constituting the independent electrode includes a solid metal element at room temperature, a conductive material composed of an inorganic non-metallic material, or a conductive material formed by doping a metal material and a non-metallic material. The selected material is non-cytotoxic and can stably exist inside cells.

[0014] Preferably, the nanodiamond is a diamond particle with a particle size in the range of 1 - 1000 nm, and the particle size is characterized by dynamic light scattering method, and at least contains one spin color center. The spin color center includes but is not limited to: nitrogen-vacancy defect color center, silicon-vacancy defect color center, germanium-vacancy defect color center, etc. The color centers with optically detected magnetic resonance effect in diamond are applicable to the spin color center described in the present invention.

[0015] Preferably, the living cell sample includes but is not limited to: HeLa, HEK293, Cos7, C6, etc. The method described in the present invention is applicable to most living cell samples.

[0016] Preferably, the preparation process of the probe includes:

[0017] Disperse the nanodiamond in a medium;

[0018] Place the tip of the nanoelectrode in the medium in which the nanodiamond is dispersed;

[0019] Connect the independent electrodes of the nanoelectrode to a signal source;

[0020] Turn on the signal source, and an electric field is generated at the tip of the independent electrode to capture the nanodiamond;

[0021] After capturing the nanodiamond, turn off the signal source and confirm that the nanodiamond does not fall off.

[0022] Preferably, a sine, square wave or triangular wave signal with a signal frequency greater than or equal to 100 kHz and a signal amplitude greater than or equal to 5 V is generated by a signal source, having one or more output channels, and the phase difference between the channels is adjusted with a resolution of 1 degree or more.

[0023] Preferably, quantum measurement schemes are applied for measurement by manipulating the nanodiamond at the probe tip. The quantum measurement schemes include but are not limited to: optically detected magnetic resonance spectroscopy, longitudinal relaxation measurement, Rabi oscillation, spin echo, or nuclear magnetic resonance, etc. Existing quantum measurement schemes are applicable to the measurement method described in the present invention.

[0024] Preferably, by measuring the spin properties of the nanodiamond at the probe tip, the measurement of multiple physical fields inside living cells is realized, including:

[0025] Calibrating the spin properties of the nanodiamond, mapping the spin properties of the probe extracted from the measurement data inside the living cell using the working curve of the spin properties of the nanodiamond, and mapping the change in the measured spin properties of the probe to the change in the measured physical quantity to obtain the measurement data of multiple physical fields in living cells.

[0026] Preferably, calibrating the spin properties of the nanodiamond includes:

[0027] Placing the probe outside the living cell, adjusting the physical quantity to be measured by the probe to a series of known values, applying the same quantum measurement scheme as that for measurement inside the living cell to the probe under each condition for measurement, analyzing the measurement data to extract the spin properties of the probe, and thus plotting the working curve of the physical quantity to be measured with respect to the spin properties of the nanodiamond as calibration data.

[0028] Compared with the prior art, the beneficial effects of the present invention at least include:

[0029] (1) The present invention is based on a nanoscale electrode to capture a nanodiamond containing a spin color center to prepare a probe, which has good biocompatibility, and the preparation method is simple and the cost is low. It can actively regulate the spatial position of the probe in living cells under the manipulation of the nanoscale electrode, and can also realize long-term continuous observation of the same site inside living cells, broadening its application scope in biomedical research.

[0030] (2) The nanodiamond probe of the present invention based on the nanoscale electrode can achieve a nanoscale spatial resolution. This probe can also calibrate the performance of the nanodiamond at the tip before and after testing, realizing the reuse of the probe and improving its practicality and economy. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 It is a schematic flow chart of a method for manipulating a single nanodiamond to measure multi-physical fields in living cells provided by an embodiment of the present invention.

[0033] Figure 2 It is a front view schematic diagram of a nanodiamond probe based on a nanoelectrode provided by an embodiment of the present invention.

[0034] Figure 3 It is a side view schematic diagram of a nanodiamond probe based on a nanoelectrode provided by an embodiment of the present invention.

[0035] Figure 4 It is the result of Embodiment 1 of the present invention: A1 and A2 are schematic diagrams before and after the carbon nanoelectrode captures the nanodiamond respectively; B1 and B2 are images observed under a fluorescence microscope before and after the carbon nanoelectrode captures the nanodiamond respectively; C is an image collected when the prepared nanodiamond probe based on the nanoelectrode simultaneously turns on bright-field illumination and fluorescence illumination; D is a scanning electron microscope image of the prepared nanodiamond probe based on the nanoelectrode.

[0036] Figure 5 It is the result of Embodiment 2 of the present invention: A-C are bright-field images of the nanodiamond probe based on the nanoelectrode at different positions in the cell; D is the result statistics of manipulating a single nanodiamond particle to perform multi-site temperature measurement in a living cell. The significance (p-value) between the data calculated by the t-test is indicated by "*", "**" indicates p < 0.01, "*" indicates p < 0.05, and "ns" indicates no significance.

[0037] Figure 6 It is the result of Embodiment 3 of the present invention: A is a schematic diagram of the change in the concentration of free radicals caused by physiological processes in the cell measured by the nanodiamond probe based on the nanoelectrode; B is the result of manipulating a single nanodiamond particle to perform multi-site free radical concentration measurement in a living cell, and the data is used to extract the longitudinal relaxation time by single-exponential decay fitting.

[0038] Figure 7 It is the result of Embodiment 4 of the present invention: A is the selection of the pulse interval in single-point longitudinal relaxation spectroscopy measurement; B is the result of manipulating a single nanodiamond particle to continuously measure the free radical concentration at a single site in a living cell for a long time.

[0039] Figure 8This is the result of Embodiment 5 of the present invention: Manipulating a single nanodiamond particle to continuously measure the concentration of free radicals at a single site within a stimulated living cell. Comparing the experimental group with the control group can verify the reliability of the measured data. Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific implementation manners described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0041] The inventive concept of the present invention is as follows: Aiming at the problem that there is a lack of a method for actively regulating the spatial position of nanodiamonds in living cells in the prior art, an embodiment of the present invention provides a method for manipulating a single nanodiamond to measure multiple physical fields in living cells, including a nanodiamond probe based on a nanoelectrode and its intracellular measurement method. The tip of the nanodiamond probe based on the nanoelectrode is loaded with a single nanodiamond containing a spin color center, and its fluorescence localization spatial resolution can reach 4.5 nm. Using the nanodiamond probe based on the nanoelectrode for intracellular measurement can achieve multi-site measurement inside living cells, long-term continuous observation of specific sites, and allows other stimuli to be applied to the cells during the observation to study the physiological behaviors of the cells. Different from the existing intracellular measurement methods based on nanodiamonds containing spin color centers, this method can actively manipulate the spatial position of nanodiamonds in living cells, allowing long-term measurement at any position inside the cells while maintaining the cell viability so that it can react normally to external stimuli.

[0042] As Figure 1 shown, the embodiment provides a method for manipulating a single nanodiamond to measure multiple physical fields in living cells, including the following steps:

[0043] S1. Capturing a nanodiamond through a nanoelectrode to form a probe, and manipulating the probe to move the nanodiamond into a living cell sample.

[0044] S2. Measuring the spin properties of the nanodiamond at the tip of the probe to achieve the measurement of multiple physical fields in the living cell.

[0045] Among them, as Figure 2 and Figure 3As shown, the nanodiamond probe based on nanoelectrodes in step S1 includes nanoelectrodes and a nanodiamond containing spin color centers at the tip of the nanoelectrodes. Among them, the nanoelectrodes include four identical independent electrodes with their roots fixed on an insulating housing for fixing and manipulating the nanodiamond. The insulating housing includes four cavities complementary in shape to the independent electrodes, enabling the roots of the independent electrodes to be installed in the cavities and the tips of the independent electrodes to be exposed, and enabling the tips of the multiple independent electrodes to be in the same plane and the connecting lines of the tip centers to form a square, the size of which is comparable to the size of the nanodiamond.

[0046] Example 1: Preparation of a nanodiamond probe based on nanoelectrodes

[0047] In Example 1, single diamond particles in a colloid were captured using carbon nanoelectrodes with an independent electrode spacing of about 100 nm. Nanodiamonds containing nitrogen-vacancy defect color centers with a particle size of 100 nm were dispersed in deionized water to form a colloid with a concentration of 0.002 mg / mL. The colloid was treated with an ultrasonic cleaner for 30 minutes to prevent agglomeration of the nanodiamonds. The carbon nanoelectrodes were fixed on a displacement stage and connected to a dual-channel signal generator through a BNC-to-Dupont wire adapter. The colloid solution was added to the sample cell of an inverted fluorescence microscope. The microscope light source was turned on, and the fluorescence filter cube was switched to green excitation light to excite the fluorescence of the nanodiamonds. The focal plane was adjusted so that the imaging focal plane was in the colloid. The carbon nanoelectrodes were moved to the center of the field of view, and at the same time, fluorescence illumination and bright-field illumination were turned on to observe the carbon nanoelectrodes and the nanodiamonds simultaneously. The dual-channel signal generator was set so that the output signal was a square wave with a frequency of 1 MHz and an amplitude of 2 Vpp, and the amplitudes, frequencies, and phases of the dual channels were synchronized. The signal output was turned on, and the nanodiamonds were captured by the trapping electric field generated at the tips of the carbon nanoelectrodes to the electrode tips. The signal output was turned off, and the nano-carbon electrodes were moved to make the probe repeatedly enter and exit the colloid to confirm that the nanodiamonds did not fall off, completing the preparation of the nanodiamond probe based on nanoelectrodes, and obtaining the results as Figure 4 shown.

[0048] Example 2: Manipulating single nanodiamond particles to perform multi-site temperature measurements inside living cells

[0049] In Example 2, a nano-diamond probe based on a nanoelectrode was used to measure the temperature of a specific region inside a living cell. HEK cells cultured to the third generation were passaged onto a glass slide with a thickness of 0.17 mm and cultured overnight until they were fully adherent. The glass slide was installed in a sample cell with a living cell culture function compatible with an inverted fluorescence microscope, an appropriate amount of culture medium was added, the culture temperature was set at 37 °C, and the sample cell was installed on the inverted microscope and cultured for 30 minutes. During this period, a pre-prepared nano-diamond probe containing nitrogen-vacancy defect color centers based on a nanoelectrode was fixed on a displacement platform compatible with an inverted fluorescence microscope, and the probe was moved so that its tip was 30 - 50 μm above the plane where the bottom of the cell was located, and then left stationary. After the culture ended, the probe was moved near the target cell, and the optically detected magnetic resonance spectrum of the nitrogen-vacancy defect color centers at the tip of the probe was collected in the culture medium; the probe was moved to the region with dense mitochondria, and the optically detected magnetic resonance spectrum of the nitrogen-vacancy defect color centers at the tip of the probe was collected; the probe was moved into the cell nucleus, and the optically detected magnetic resonance spectrum of the nitrogen-vacancy defect color centers at the tip of the probe was collected; the probe was moved into the cytoplasm, and the optically detected magnetic resonance spectrum of the nitrogen-vacancy defect color centers at the tip of the probe was collected; the probe was moved outside the cell, the temperature of the sample cell was adjusted, and the optically detected magnetic resonance spectra of the nitrogen-vacancy defect color centers at the tip of the probe at a series of temperatures were collected. The above measurement steps were repeated to measure multiple cells, the obtained optically detected magnetic resonance spectra were fitted, the peak frequencies were extracted, and the actual measured temperature was calculated by combining the working curves obtained by collecting at a series of temperatures. The measurement data were statistically analyzed to obtain the results as shown in Figure 5 shown.

[0050] Example 3: Manipulating single nano-diamond particles to measure the multi-site free radical concentration inside living cells

[0051] In Example 3, a nano-diamond probe based on a nanoelectrode was used to measure the free radical concentration in a specific region within a living cell. C6 cells cultured to the third generation were passaged onto a glass slide with a thickness of 0.17 mm and cultured overnight to allow them to adhere completely. The glass slide was mounted in a sample cell with a live cell culture function compatible with an inverted fluorescence microscope, an appropriate amount of culture medium was added, the culture temperature was set at 37 °C, and the sample cell was mounted on the inverted microscope and cultured for 30 minutes. During this period, a pre-prepared nano-diamond probe containing a nitrogen-vacancy defect color center based on a nanoelectrode was fixed on a displacement platform compatible with an inverted fluorescence microscope, and the probe was moved so that its tip was 30 - 50 μm above the plane where the bottom of the cell was located, and then left stationary. After the culture ended, the probe was moved near the target cell, and the spin longitudinal relaxation spectrum of the nitrogen-vacancy defect color center at the tip of the probe was collected in the culture medium; the probe was moved into the cell nucleus, and the spin longitudinal relaxation spectrum of the nitrogen-vacancy defect color center at the tip of the probe was collected; the probe was moved into the cytoplasm, and the spin longitudinal relaxation spectrum of the nitrogen-vacancy defect color center at the tip of the probe was collected. The obtained spin longitudinal relaxation spectra were fitted to extract the spin relaxation time. Since the spin relaxation rate of the nitrogen-vacancy defect color center is related to the free radical concentration in the environment, the obtained spin relaxation time can reflect the difference in free radical concentrations at different sites in the cell, and the results are as shown in Figure 6 shown.

[0052] Example 4: Manipulating a single nano-diamond particle to continuously measure the free radical concentration at a single site within a living cell for a long time

[0053] In Example 4, a nano-diamond probe based on a nanoelectrode was used to continuously measure the free radical concentration at a single site inside living cells for a long time. C6 cells cultured to the third generation were passaged onto a glass slide with a thickness of 0.17 mm and cultured overnight until they were completely adherent. The glass slide was installed in a sample cell with a live cell culture function compatible with an inverted fluorescence microscope, an appropriate amount of culture medium was added, the culture temperature was set to 37 °C, and the sample cell was installed on the inverted microscope and cultured for 30 minutes. During this period, a pre-prepared nano-diamond probe containing a nitrogen-vacancy defect color center based on a nanoelectrode was fixed on a displacement platform compatible with an inverted fluorescence microscope, and the probe was moved so that its tip was 30 - 50 μm above the plane where the bottom of the cell was located, and then left stationary. After the culture was completed, the probe was moved near the target cell, and the spin longitudinal relaxation spectrum of the nitrogen-vacancy defect color center at the tip of the probe was collected in the culture medium, and the pulse interval corresponding to when the fluorescence contrast decayed to 37% of the maximum value was extracted. The probe was moved to the region of interest inside the target cell, the pulse interval time was fixed, single-point spin longitudinal relaxation data was collected, the sampling interval was set to 180 ms, and the recording continued for 1 - 2 hours. In the control experiment, the probe was moved outside the target cell, the pulse interval time was fixed, single-point spin longitudinal relaxation data was collected, the sampling interval was set to 180 ms, and the recording continued for 1 - 2 hours. Long-term continuous measurements were performed on different regions of the cells, the data was filtered and downsampled, and the data characteristics were compared to confirm that the collected signal did not come from system noise, and the results as shown in Figure 7 were obtained.

[0054] Example 5: Manipulating a single nano-diamond particle to continuously measure the free radical concentration at a single site inside a stimulated living cell for a long time

[0055] In Example 5, a nano-diamond probe based on a nanoelectrode was used to continuously measure the concentration of free radicals at a single site in a stimulated living cell for a long time. C6 cells cultured to the third generation were passaged onto a glass slide with a thickness of 0.17 mm and cultured overnight to ensure complete adhesion. The glass slide was installed in a sample cell with a live cell culture function compatible with an inverted fluorescence microscope, an appropriate amount of culture medium was added, the culture temperature was set to 37 °C, and the sample cell was installed on the inverted microscope and cultured for 30 minutes. During this period, a pre-prepared nano-diamond probe containing a nitrogen-vacancy defect color center based on a nanoelectrode was fixed on a displacement platform compatible with an inverted fluorescence microscope. The probe was moved so that its tip was 30 - 50 μm above the plane where the bottom of the cell was located, and then it was left stationary. After the culture ended, the probe was moved near the target cell, and the spin longitudinal relaxation spectrum of the nitrogen-vacancy defect color center at the tip of the probe was collected in the culture medium. The pulse interval corresponding to when the fluorescence contrast decayed to 37% of the maximum value was extracted. The probe was moved to the region of interest within the target cell, the pulse interval time was fixed, and single-point spin longitudinal relaxation data was collected. The sampling interval was set to 180 ms, and the recording continued for 1 - 2 hours. During this period, glucose or 2-deoxy-D-glucose was added to make the final concentrations 13.5 g / L or 10 mM respectively. In the control experiment, the probe was moved into the culture medium, the pulse interval time was fixed, single-point spin longitudinal relaxation data was collected, the sampling interval was set to 180 ms, and the recording continued for 1 - 2 hours. During this period, glucose or 2-deoxy-D-glucose was added to make the final concentrations 13.5 g / L or 10 mM respectively. Long-term continuous measurements were performed on different regions of the cells, the data was filtered and downsampled, and the data characteristics were compared to confirm that the collected signals did not come from the added reagents, and the results were as Figure 8 shown.

[0056] In summary, a method for manipulating a single nano-diamond to measure multiple physical fields in a living cell provided in the embodiment of the present invention uses a nano-diamond probe based on a nanoelectrode for measurement inside a living cell, has high spatial controllability, time continuity, and compatibility with cell physiological activities, provides a powerful tool for understanding the physical and chemical processes inside cells and their interaction mechanisms, and is expected to promote research progress in the fields of cell biology, biomedicine, and nanomedicine.

[0057] The above-described specific embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for manipulating a single nanodiamond to measure multi-physical fields in living cells, characterized in that, Including the following steps: Forming a probe by capturing a nanodiamond with a nanoelectrode, and manipulating the probe to move the nanodiamond into a living cell sample; Measuring multiple physical fields in a living cell by measuring the spin properties of the nanodiamond at the tip of the probe.

2. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 1, wherein The nanoelectrode includes a plurality of independent electrodes with roots fixed on an insulating housing, and the nanodiamond is captured and manipulated through the plurality of independent electrodes.

3. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 2, wherein The insulating housing includes a plurality of cavities complementary in shape to the independent electrodes, enabling the roots of the independent electrodes to be installed in the cavities and the tips of the independent electrodes to be exposed, and enabling the tips of the plurality of independent electrodes to be in the same plane and the connecting lines of the tip centers to form a regular polygon, the size of the regular polygon being comparable to the size of the nanodiamond.

4. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 2, wherein The cross-section of each independent electrode gradually decreases from the root to the tip, the tip being a sphere with a radius of curvature of 1 - 1000 nm, and the material constituting the independent electrode includes a metallic element that is solid at room temperature, a conductive material composed of an inorganic non-metallic material, or a conductive material formed by doping a metallic material and a non-metallic material.

5. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 1, wherein The nanodiamond is a diamond particle with a particle size of 1 - 1000 nm, which contains at least one spin color center.

6. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to any one of claims 2-5, characterized in that, The preparation process of the probe includes: Dispersing the nanodiamond in a medium; Placing the tip of the nanoelectrode in the medium in which the nanodiamond is dispersed; Connecting the independent electrodes of the nanoelectrode to a signal source; Turning on the signal source, and an electric field is generated at the tip of the independent electrode to capture the nanodiamond; After capturing the nanodiamond, turning off the signal source and confirming that the nanodiamond does not fall off.

7. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 6, wherein Generating a sine, square wave or triangular wave signal with a signal frequency greater than or equal to 100 kHz and a signal amplitude greater than or equal to 5 V through the signal source, having one or more output channels, and adjusting the phase difference between the channels with a resolution of 1 degree or more.

8. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 1, characterized in that, Performing a measurement by applying a quantum measurement scheme by manipulating the nanodiamond at the tip of the probe, and the quantum measurement scheme includes optically detected magnetic resonance spectroscopy, longitudinal relaxation measurement, Rabi oscillation, spin echo or nuclear magnetic resonance.

9. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 1, wherein Measuring multiple physical fields in a living cell by measuring the spin properties of the nanodiamond at the tip of the probe, including: Calibrating the spin properties of the nanodiamond, mapping the spin properties of the probe measured in the living cell using the working curve of the spin properties of the nanodiamond, and mapping the change in the spin properties of the probe measured to the change in the measured physical quantity to obtain the measurement data of the multiple physical fields of the living cell.

10. The method for manipulating a single nanodiamond to measure multi-physical fields in living cells according to claim 9, wherein, Calibrating the spin properties of the nanodiamond includes: Placing the probe outside the living cell, adjusting the physical quantity to be measured by the probe to a series of known values, performing a measurement on the probe under each condition by applying the same quantum measurement scheme as that measured in the living cell, analyzing the measurement data to extract the spin properties of the probe, and thus plotting the working curve of the physical quantity to be measured with respect to the spin properties of the nanodiamond as calibration data.

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