Cytoplasm viscosity sensor and preparation method thereof

Abstract

The invention discloses a cytoplasm viscosity sensor and a preparation method thereof. The preparation method of the cytoplasm viscosity sensor comprises the following steps: drawing a silicon borate capillary tube into a micro / nano suction tube with the inner diameter of 120-1300 nm; and injecting the prepared phosphate buffer solution into the prepared micro / nano suction tube to complete the preparation of the cytoplasm viscosity sensor. The cytoplasm viscosity sensor can be prepared by pouring a low-viscosity phosphate buffer solution into the drawn nano suction tube. The method is simple, efficient, low in dependence on equipment, free of by-products, high in success rate and good in repeatability, and the prepared nanopipette viscosity sensor is sensitive in viscosity response and high in response speed and can be used for measuring cytoplasm viscosity.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a cytoplasmic viscosity sensor and a preparation method thereof. Background Art

[0002] Cytoplasmic viscosity is an important cytoplasmic microenvironment parameter and plays an important role in various biological processes in cells. Abnormal cytoplasmic viscosity not only affects these physiological processes, but is also closely related to the occurrence and development of diseases. The development of cytoplasmic viscosity sensors is of great value for understanding viscosity-related pathological processes and early diagnosis of diseases.

[0003] Nanostraws refer to tubular objects with a hollow structure at the nanoscale and a tip opening. They are usually made of quartz / borosilicate capillaries pulled by a microelectrode puller. Unlike traditional straws used to transfer liquids, nanostraws are widely used in single-cell experiments such as intracellular injection, cell biopsy, and intracellular sensing due to their nano-sized tips, hollow structures, and ion transport behaviors that can respond to external stimuli. In 2021, researchers developed a method for measuring cytoplasmic viscosity using double nanopipette. However, the use of double nanostraws increases the difficulty of operation and the risk of cell damage. In addition, double nanostraws measure the average viscosity of the cytoplasm between the two nanostraws, which makes it difficult to accurately determine the viscosity differences between the subregions of the cytoplasm. In order to solve these problems, a θ-type nanostraw viscometer was developed in 2022, which effectively reduced the interference of the distance between the two nanostraws on the measurement results. However, this method still relies on double-hole nanostraws to detect cytoplasmic viscosity. Compared with single-hole nanostraws, θ-type nanostraws are difficult to manufacture, which brings greater technical challenges to the manufacture of θ-type nanostraws. In addition, both of the above methods rely on the intensity of ionic current to measure solution viscosity. In the cytoplasmic ion concentration range, the sensitivity of ionic current to changes in ion concentration is higher than that of ionic current rectification (ICR). Therefore, the development of a single nanopore nanostraw cytoplasmic viscosity sensor based on ICR can provide a simple, stable and reliable tool for viscosity measurement. Summary of the invention

[0004] The present invention provides a cytoplasm viscosity sensor and a preparation method thereof to solve the above problems.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] A method for preparing a cytoplasmic viscosity sensor comprises the following steps:

[0007] S1: Pull borosilicate capillaries into micro / nano pipettes with an inner diameter of 120nm-1300nm;

[0008] S2: Inject the prepared phosphate buffer into the micro / nano pipette prepared in step S1 to complete the preparation of the cytoplasmic viscosity sensor.

[0009] When the drawing parameters are Heat = 350, Fil = 0, Vel = 20, Del = 145, Pull = 160, the inner diameter of the obtained nanostraw is about 120nm; when the drawing parameters are Heat = 300, Fil = 0, Vel = 20, Del = 145, Pull = 160, the inner diameter of the obtained nanostraw is about 150nm; when the drawing parameters are Heat = 275, Fil = 0, Vel = 20, Del = 145, Pull = 160, the inner diameter of the obtained nanostraw is about 200nm; when the drawing parameters are Heat = 230, Fil = 0, Vel = 20, Del = 145, Pull = 160, the inner diameter of the obtained nanostraw is about 340nm; When the drawing parameters are Heat=210, Fil=0, Vel=20, Del=145, Pull=160, the inner diameter of the prepared nanostraw is about 460nm; when the drawing parameters are Heat=200, Fil=0, Vel=20, Del=145, Pull=160, the inner diameter of the prepared nanostraw is about 660nm; when the drawing parameters are Heat=200, Fil=0, Vel=20, Del=145, Pull=130, the inner diameter of the prepared nanostraw is about 900nm; when the drawing parameters are Heat=220, Fil=0, Vel=18, Del=145, Pull=120, the inner diameter of the prepared nanostraw is about 1300nm.

[0010] Furthermore, in S2, the buffer solution is a phosphate buffer solution, and its viscosity at 25°C is 0.91 mPa.s.

[0011] Furthermore, the phosphate buffer solution is composed of 0.8 mM K 2 HPO 4 , 0.2 mM KH 2 PO 4 The pH value of the solution was prepared with 100 mM KCl to 7.4.

[0012] Furthermore, in S1, the inner diameter of the borosilicate capillary is 0.78 mm and the outer diameter is 1.0 mm.

[0013] Furthermore, in S1, the drawing parameters are Heat=200-350, Fil=0, Vel=18-20, Del=145, Pull=120-160.

[0014] A cytoplasm viscosity sensor is prepared by the preparation method of the cytoplasm viscosity sensor.

[0015] The beneficial effects of the present invention are:

[0016] The invention discloses a method for preparing a cytoplasm viscosity sensor, which can be prepared by injecting a low-viscosity phosphate buffer into a drawn nanopipette. The method is simple, efficient, has low equipment dependence, has no by-products, and has a high success rate and good repeatability. The prepared nanopipette viscosity sensor has a sensitive response to viscosity and a fast response speed, and can measure cytoplasm viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Photos of prepared micro / nano straws with different inner diameters (120-1300nm);

[0019] Figure 2 This is a graph showing the solution viscosity response behavior of a nanopipette viscosity sensor with a pore size of 150 nm based on ionic current rectification;

[0020] Figure 3 The viscosity response working curve of the nanopipette viscosity sensor with an pore size of 150 nm based on the rectification ratio (n=6);

[0021] Figure 4 The IV curves of the reversible response of the nanopipette viscosity sensor with a pore size of 150 nm to two viscosity solutions of 0.91 and 40.07 mPa.s (n=6);

[0022] Figure 5 The rectification ratio oscillation diagram of the reversible response of the nanopipette viscosity sensor with an pore size of 150 nm to two viscosity solutions of 0.91 and 40.07 mPa.s (n=6);

[0023] Figure 6 The effect of different concentrations of KCl on the viscosity response sensitivity of the nanopipette viscosity sensor with a pore size of 150 nm (n=6);

[0024] Figure 7 The effect of different solution pH on the viscosity response sensitivity of the nanopipette viscosity sensor with a pore size of 150 nm (n=6);

[0025] Figure 8 The effect of different pore sizes on the viscosity response sensitivity of the nanostraw viscosity sensor (n=6);

[0026] Fig. 9 This is a microscopic picture of a 150nm pore size nanopipette viscosity sensor piercing a cell to measure viscosity;

[0027] Fig.10 The ion current rectification curve of the cytoplasmic viscosity change of the nanopipette viscosity sensor with an pore size of 150 nm before and after high concentration salt solution stress;

[0028] Fig.11 This is a statistical diagram of the rectification ratio of the changes in cytoplasmic viscosity of the nanopipette viscosity sensor with an pore size of 150 nm before and after high concentration salt solution stress (n=6). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example:

[0031] Borosilicate capillaries with an outer diameter of 1.0 mm and an inner diameter of 0.78 mm were drawn into nanopipette tubes with an inner diameter of 150 nm using a P-2000 microelectrode laser drawing instrument. The drawing parameters were Heat = 300, Fil = 0, Vel = 20, Del = 145, and Pull = 160. A 0.1 mM phosphate buffer (composed of 0.8 mM K 2 HPO 4 , 0.2 mM KH 2 PO 4 and 100 mM KCl prepared with a viscosity of 0.91 mPa.s), and then the prepared phosphate buffer was injected into the nano-straw with an inner diameter of 150 nm through a micropipette, and then placed in a centrifuge tube filled with the above phosphate buffer for storage.

[0032] Performance testing:

[0033] The ion transport behavior characteristics of the nanopipette were tested by the electrochemical workstation CHI830D. First, a small amount of solution was used to soak the end of the capillary, and then the solution was injected into the capillary with a micro-syringe to prevent bubbles from being generated during the perfusion. An Ag / AgCl reference electrode with a diameter of 0.3 mm was inserted into the tail end of the nanocapillary as the working electrode, and another Ag / AgCl reference electrode was inserted into the electrolytic cell buffer as the counter electrode and reference electrode. The linear sweep voltammetry method (-1.0V-+1.0V, 0.1V / s) was used to record the ion current, and the data was recorded after stabilization. The rectification ratio r (r = log 2 |I+ / I-|) is used to quantify the direction and strength of ICR, where I+ and I- represent the ion current under +1V and -1V bias voltages, respectively. When I+>I-, r is positive, and when I+<I-, r is negative. The positive or negative value of r represents the direction of ICR, and the absolute value of r represents the strength of ICR. The viscosity response sensitivity of the viscosity response rectification ratio difference of 0.91mPa.s and 40.07mPa·s solutions is obtained by comparing the viscosity change of the solution, and is expressed as (r 40.07mPa.s -r 0.91mPa.s ) / (40.07mPa.s-0.91mPa.s).

[0034] Take T98G cells in the logarithmic growth phase and use a microscope, micro-ultrasound system (MP-365) and electrochemical workstation (CHI830D) to detect the intracellular ion current of single cells. After the micro / nano-pipette cytoplasmic viscosity probe is inserted into the cell, slowly add NaCl solution to make the final concentration of NaCl 290mM. Put a silver / silver chloride electrode (0.3mm) into the culture dish solution as the counter electrode and reference electrode, and insert another silver / silver chloride electrode into the lumen from the rear end of the micro / nano-pipette as the working electrode. Use IV mode to record the changes in cell cytoplasmic viscosity before and after the addition of NaCl.

[0035] from Figure 1 It can be seen that by adjusting the drawing parameters, a series of micro / nano straws with different diameters (120-1300nm) can be drawn.

[0036] from Figure 2 It can be seen that the 150 nm diameter nanostraw viscosity sensor exhibits IV curves with different ICR intensities in solutions with different viscosities, indicating that the nanostraw viscosity probe has viscosity response capability based on ICR.

[0037] from Figure 3As can be seen from the figure, the rectification ratio of the 150 nm diameter nanopipette viscosity sensor first increases and then saturates with the increase of solution viscosity. The linear fitting in the inset shows that in the viscosity range of 0.91 mPa·s to 40.07 mPa·s, the rectification ratio of the micro / nano-pipette ICR response has a good linear relationship with the solution viscosity (r = 0.14μ-0.20; R 2 =0.919; n=6).

[0038] from Figure 4 It can be seen that the 150nm diameter nanostraw viscosity sensor has different ICR directions in the two viscosity solutions of 0.91mPa·s and 40.07mPa·s. The ICR direction changes repeatedly by repeatedly changing the solution viscosity, and they overlap well in solutions of the same viscosity, indicating that the nanostraw viscosity sensor has good reversibility of viscosity response.

[0039] from Figure 5 It can be seen that the nanostraw viscosity sensor with a diameter of 150 nm has different rectification ratios in the two viscosity solutions of 0.91 and 40.07 mPa·s. The rectification ratio oscillates repeatedly by repeatedly changing the solution viscosity, indicating that the nanostraw viscosity sensor has good reversibility of viscosity response.

[0040] from Figure 6 It can be seen that with the increase of solution salt concentration, the viscosity response sensitivity of the 150nm diameter nanostraw viscosity sensor first increases and then weakens, showing the best sensitivity near 100mM.

[0041] from Figure 7 It can be seen that as the pH value of the solution increases, the viscosity response sensitivity of the 150 nm diameter nanostraw viscosity sensor gradually increases.

[0042] from Figure 8 It can be seen that as the diameter of the micro / nano straw increases, the viscosity response sensitivity of the micro / nano straw viscosity sensor gradually weakens, and the nano straw viscosity sensor with a pore size less than 150nm has a better viscosity response sensitivity.

[0043] from Fig. 9 As can be seen, after the 150 nm diameter nanopipette viscosity sensor was inserted into the cell, the cell morphology did not change significantly, indicating that the nanopipette is a probe with low cell loss.

[0044] from Fig.10 It can be seen that after adding 290mM NaCl to T98G cells, the ICR direction of the 150nm diameter nanopipette viscosity sensor reversed. This test result shows that the cytoplasmic viscosity increases in a high salt environment. This is consistent with the research report that a high salt environment promotes the increase of cytoplasmic viscosity.

[0045] from Fig.11 It can be seen that the rectification ratios of six independent T98G cells increased to varying degrees after adding NaCl, which indicates that the nanostraw viscosity sensor prepared by the present invention is a reliable viscosity sensor based on ICR response.

[0046] The present invention discloses a method for preparing a single-cell intracellular viscosity sensor based on a nanopipette, wherein a phosphate buffer solution is injected into a drawn micro / nano-pipette to prepare the nanopipette intracellular viscosity sensor. The method is simple, efficient, has low equipment dependence, has no by-products, and has a high success rate and good repeatability. The prepared nanopipette viscosity sensor is sensitive to viscosity and has a fast response speed, and can perform real-time monitoring and continuously record the dynamic changes of viscosity in a single cell.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a cytoplasmic viscosity sensor, characterized in that: The steps include: S1: Pull borosilicate capillaries into micro / nano pipettes with an inner diameter of 120nm-1300nm; S2: Inject the prepared buffer into the micro / nano pipette prepared in step S1 to complete the preparation of the cytoplasmic viscosity sensor.

2. The method for preparing the cytoplasmic viscosity sensor according to claim 1, characterized in that: In S2, the buffer solution is a phosphate buffer solution, and its viscosity at 25°C is 0.91 mPa.s.

3. The method for preparing the cytoplasmic viscosity sensor according to claim 2, characterized in that: The phosphate buffer solution is a solution prepared from 0.8 mM K2HPO4, 0.2 mM KH2PO4 and 100 mM KCl with a pH value of 7.

4.

4. The method for preparing the cytoplasmic viscosity sensor according to claim 1, characterized in that: In S1, the inner diameter of the borosilicate capillary is 0.78 mm and the outer diameter is 1.0 mm.

5. The method for preparing the cytoplasmic viscosity sensor according to claim 1, characterized in that: In S1, the drawing parameters are Heat = 200-350, Fil = 0, Vel = 18-20, Del = 145, Pull = 120-160.

6. A cytoplasmic viscosity sensor, characterized in that: The cytoplasmic viscosity sensor is prepared by the preparation method of claim 1.

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