A method for measuring surface charge of a biomaterial using a bubble probe

Abstract

The application provides a method for measuring the surface charge of biological materials by using a bubble probe, and belongs to the technical field of physical chemistry instrument analysis. The method comprises the following steps: sticking an isosceles right prism with a back plated layer on a fluid force Holder by using AB glue, then assembling the fluid force probe, immersing the probe into water after applying a certain positive pressure, realizing real-time observation and size measurement of the bubble by providing a lateral light source, then measuring the force curve under the set parameters by using software, and judging the charging condition of the biological materials by the adhesion force between the bubble and the biological materials. The application realizes the generation of a stable micron-level bubble with controllable size at the front end of the probe by using a fluid force device and a lateral light path system, and can measure the surface charging condition of the biological materials by using the bubble, and has the advantages of rapidness, simplicity, in-situ measurement and small damage.

Description

Technical Field

[0001] This invention relates to a method for measuring the surface charge of biological materials using a bubble probe, belonging to the field of physical and chemical instrument analysis technology. Background Technology

[0002] Surface charge is widely present on the surface of biomaterials and is closely related to many dynamic equilibria and physiological processes. Its sign and density largely determine the surface properties. For example, cell surface charge is mainly determined by the lipid bilayer and protein charge of the cell membrane, and it can vary depending on species, cell type, and differentiation state. It has been widely confirmed that cell surface charge affects many membrane-regulated cellular functions, such as endocytosis, myocyte contraction, nutrient transport, and hormone release. On the other hand, cell surface charge can also be used as a marker for cell analysis and diagnosis. Articles have reported changes in cell surface charge of various types of stem cells during differentiation, as well as differences between cancer cells and normal cells. Therefore, the ability to identify the surface charge of different types of living cells and to visualize local cell surface charge is of great significance for understanding many fundamental cellular functions. However, the measurement and mapping of biomaterial surface charge remains a significant challenge due to the lack of robust techniques capable of performing micro- and nanoscale measurements in complex environments. Traditional techniques such as zeta potential measurement or potentiometric titration cannot provide information on charge distribution at biomaterial interfaces, nor can they measure the charge of tiny materials such as single cells. Recently, scanning ion conductivity microscopy has been used for mapping biomaterial surface charges. This method is based on the principle of ion current rectification and uses a single nanopipette to measure sample morphology and surface charge density. However, the nanopipette needs to first approach the edge of the sample and then return to a neutral position through trial and error. This process is cumbersome and requires a very long time to scan the sample surface point by point. Therefore, this technique cannot perform rapid surface charge characterization.

[0003] Hydrodynamic microscopy (HFM) is an atomic force microscope based on a hollow cantilever, enabling fluid dispensing and stimulation of single live cells under physiological conditions. Since its inception in 2009, HFM has become one of the most promising tools for biological research at the micro- and nanoscale. HFM combines the precise force control of AFM with the versatility of microfluidics, integrating micro-channels into the AFM cantilever and connecting them to a vacuum pump via an injection tube. This forms a continuous and closed channel that can be filled with any chosen solution and immersed in a liquid environment. A small orifice at the AFM needle tip at the cantilever end allows for fluid dispensing and cell manipulation, primarily for pipetting; however, there are no reports of charge measurement applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for measuring the surface charge of different biological materials by generating stable bubbles at the tip of a probe using a hydrodynamic microscope. In a typical liquid environment, the bubble surface carries a weak negative charge. The adhesion force between the bubble and different biological materials is primarily determined by the magnitude of the electrostatic force. By measuring the adhesion force, the magnitude of the electrostatic force between the bubble and the sample can be determined, thus revealing the surface charge of the sample. The bubbles generated by this hydrodynamic microscope probe are stable and controllable in size, much smaller than those of ordinary probes. They remain stably at the probe tip without easily detaching, preventing damage to the sample surface and making them ideal for detecting the surface charge of biological samples. Using this probe in conjunction with atomic force microscopy to detect the surface charge of biological materials offers wide applicability to various samples, a simple scanning process, short scanning time, and high accuracy despite significant differences in detection results between different charges. It fully leverages the advantages of bubble probes and has broad application prospects in physical chemistry, biology, and medicine.

[0005] The technical solution of the present invention is as follows:

[0006] A method for measuring the surface charge of biomaterials using a bubble probe includes the following steps:

[0007] (1). Optical path construction: The isosceles right-angle prism with aluminum-coated bevel is connected to the bright surface of the fluid force support Holder with AB glue; a fluid force probe is set on one side of the prism, and the bevel of its coating should face the position of the probe. A light source is set on the other side of the fluid force probe. The optical path reaches the bevel of the isosceles right-angle prism through the fluid force probe; during operation, a light source is provided on the side of the prism. The light passes through the probe and is refracted by the prism into the objective lens, so that the side image of the bubble probe can be obtained.

[0008] (2). Probe generation: Install the fluid force probe onto the fluid force holder in step (1) and connect it to the fluid force device. After applying a certain positive pressure, immerse it in the liquid. The size of the bubble at the tip can be calculated from the isosceles right-angle prism by using the light source provided from the side. Adjust the pressure to stabilize the bubble at a suitable size, which can be used for further measurement. Before immersing the probe in the liquid, a certain positive pressure must be applied to the probe. Otherwise, the liquid will fill the probe tip due to capillary action and the bubble will not be generated.

[0009] According to a preferred embodiment of the present invention, step (1) includes one or more of the following schemes:

[0010] a. The right-angle side of the isosceles right-angle prism should be 1mm. A prism that is too small is difficult to operate and more expensive, while a prism that is too large will cause the optical path to become longer, making it impossible for the objective lens to focus on the tip of the probe.

[0011] b. The reflectivity of the inclined surface of an isosceles right-angle prism coated with aluminum is 92%.

[0012] c. The connection between the fluid force support Holder and the isosceles right angle prism is to use AB glue for bonding. After applying the glue, let it stand for 24 hours to fully fix the prism.

[0013] d. The isosceles right-angle prism should be about 1 mm away from the probe. If the distance is too far, the optical path will become longer, making it impossible for the objective lens to focus on the tip of the probe.

[0014] According to a preferred embodiment of the present invention, in step (2), the hydrodynamic probe is a Micropipette probe with an opening diameter of 8 μm at the tip.

[0015] According to a preferred embodiment of the present invention, in step (2), adjusting the pressure to 250 mbar can generate suitable bubbles. At this time, the bubble radius is about 4.1 μm. Excessive pressure will cause the bubbles to become unstable and escape.

[0016] According to a preferred embodiment of the present invention, in step (2), the method for calculating the bubble radius is to move the probe 5 μm each time in the liquid, and calculate the actual size of the bubble by the ratio of the actual moving distance in the image to the bubble size.

[0017] (3) Sample preparation: The biomaterial includes cells or phospholipid bilayers. If it is a cell, proceed directly to step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid, DPPE phospholipid, or DPPG phospholipid in chloroform to form a lipid solution. First, dispense the above solution into equal amounts in glass bottles and dry them overnight in a vacuum drying oven to form a lipid film. Then, add culture buffer to the bottle to resuspend the lipid film and obtain a multilayer vesicle solution. Place the solution in a water bath sonicator at room temperature to obtain a small monolayer vesicle solution. Then, cut a mica sheet with Scotch tape to obtain a fresh surface and deposit the vesicle solution onto the mica sheet to obtain a lipid sample. After culturing the lipid sample above the phase transition temperature, cool it to room temperature and rinse it with imaging buffer. Finally, use imaging buffer to preserve the prepared sample to prevent drying.

[0018] (4). Sample measurement: After the sample is prepared, it is fixed on the sample stage of the hydrodynamic microscope and measured in the imaging buffer environment. During the measurement, no air bubbles are generated at the tip of the needle during the needle insertion process. After the needle insertion is completed, the piezoelectric ceramic is raised to the highest point and restored to normal pressure. Force curve test is started to obtain the adhesion force between the air bubble and different phospholipid samples. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the air bubble and the phospholipid is obtained, and then the charge status of the sample surface is obtained. When the adhesion force is 230 nN or above, the biomaterial is judged to be positively charged. When the adhesion force is 4 nN-10 nN, the biomaterial is judged to be neutral and uncharged. When the adhesion force is 0 nN, the biomaterial is judged to be negatively charged.

[0019] The adhesion between phospholipids and air bubbles is composed of van der Waals forces, capillary forces, and electrostatic forces. Van der Waals forces are relatively small and have little impact. Capillary forces are mainly related to the surface tension of the solution and have essentially the same value in the same liquid environment. However, micro- and nano-sized air bubbles carry a weak negative charge in a typical aqueous environment. Therefore, the adhesion between phospholipids and air bubbles is mainly determined by the electrostatic force between them. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the air bubbles and phospholipids can be determined, thereby revealing the charge on the sample surface.

[0020] According to a preferred embodiment of the present invention, step (3) includes one or more of the following schemes:

[0021] I. The concentration of the lipid solution formed is 1 mg·mL⁻¹, and the lipid membrane is resuspended to a final concentration of 0.5 mg·mL⁻¹;

[0022] II. The ultrasonic time for the water bath ultrasonic instrument is 0.5 hours;

[0023] III. The mica sheet has a diameter of 1 cm, and the vesicle solution deposited on the mica sheet is 10 μL;

[0024] IV. The DPTAP and DPPG samples were incubated at 60℃, and the DPPE samples were incubated at 70℃ for 0.5h.

[0025] V. The culture buffer consists of 10 ml HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 150 ml NaCl, and 5 ml CaCl2, with a pH of 7.3.

[0026] VI. The imaging buffer consisted of 10 ml HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 150 ml NaCl, and a pH of 7.0.

[0027] According to a preferred embodiment of the present invention, in step (4), the pressure should be reduced during the needle insertion process. The pressure during the needle insertion process is 100 mbar. At this time, no bubbles are generated at the needle tip, thereby avoiding contamination and disturbance of bubbles during the needle insertion process. After the needle insertion is completed, the piezoelectric ceramic is raised to the highest position, and the pressure is restored to 250 mbar.

[0028] According to a preferred embodiment of the present invention, in step (4), during the force curve test, the pressure is set to 10 nN, the moving distance is 15 μm, and the moving speed is 1 μm / s.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. This invention provides a novel method for manufacturing bubble probes. Traditional bubble probes involve attaching a hydrophobically treated gold sheet to the tip of a tipless cantilever, carefully injecting air bubbles into a liquid cell using an ultra-sharp pipette, and then slowly lowering the cantilever to pick up suitable bubbles using hydrophobic interactions. However, bubble probes produced by this method often have relatively large bubbles, with a radius of around 100 μm. When measuring smaller samples, they are easily affected by substrate interference, and when measuring samples with high adhesion, the bubbles tend to detach from the probe tip, making it impossible to obtain accurate adhesion data. Furthermore, because gases dissolve slowly in water, the bubble size is not constant and decreases over time, causing deviations in experimental results. The bubble probe obtained by this invention using hydrodynamic microscopy produces stable and controllable bubbles, much smaller than those in previous bubble probes. These bubbles remain stably attached to the probe tip, preventing detachment and avoiding damage to the sample surface, making it ideal for detecting biological sample surfaces.

[0031] 2. This invention provides a method for constructing an optical path for observing the tip of a hydrodynamic probe from the side. Hydrodynamic microscopy, a promising instrument in the field of biology, typically uses inverted and overhead microscopes, allowing observation of the upper and lower surfaces of the probe, but not the side, thus hindering real-time analysis. We provide a lateral optical path system where an externally supplied light source is refracted through a prism into the objective lens, enabling real-time observation of the probe's state from the side. This invention achieves real-time observation and size measurement of the bubble at the probe tip through this lateral optical path system, ensuring the accuracy of experimental results.

[0032] 3. This invention prepares large-area phospholipid bilayers with different charges using mica as a substrate. First, multilayer phospholipid vesicles are formed, then small monolayer vesicles are obtained using an ultrasonic method. Heating causes the vesicles to rupture and spread onto the mica, forming a stable phospholipid bilayer. Unlike traditional methods that typically produce small patches of phospholipid, this invention achieves this by controlling the phospholipid solution concentration, incubation temperature, ultrasonic time, and Ca2+. 2+ The concentration was adjusted to obtain a uniform phospholipid sheet covering the mica surface. This sheet has a large area and is applicable to phospholipids with different charge types, which can simulate the charge situation on the surface of various biomaterials.

[0033] 4. This invention provides a method for measuring the surface charge of biomaterials using a bubble probe. Taking a phospholipid bilayer as an example, it simulates the surface of biomaterials under different charge conditions. In a typical liquid environment, the bubble surface carries a weak negative charge. The magnitude of the adhesion force between the bubble and different biomaterials is mainly determined by the magnitude of the electrostatic force. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the bubble and the sample can be determined, thereby revealing the charge status of the sample surface. Experimental tests have shown that the adhesion force between phospholipids with different charges and bubbles varies significantly. This method has advantages such as wide applicability to various samples, simple scanning operation, short scanning time, and high detection accuracy. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the side optical path construction;

[0035] The components are: 1. Light source, 2. Fluid force support, 3. Isosceles right angle prism, 4. Sample, 5. Objective lens, 6. Probe;

[0036] Figure 2 This is a schematic diagram of a bubble probe obtained under appropriate pressure.

[0037] Figure 3 This is a schematic diagram of a large-area DPTAP phospholipid bilayer on the surface of mica;

[0038] Figure 4 This is a schematic diagram of a large-area DPPE phospholipid bilayer on the surface of mica.

[0039] Figure 5 This is a schematic diagram of a large-area DPPG phospholipid bilayer on the surface of mica;

[0040] Figure 6 Without adding Ca 2+ A schematic diagram of the isolated island-like structure of the formed phospholipid bilayer;

[0041] Figure 7 Box plots show the adhesion forces between three different charged phospholipids and air bubbles;

[0042] Figure 8 Box plots show the adhesion force data between mica and air bubbles, and the adhesion force data between phospholipids and air bubbles. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments and accompanying drawings, but is not limited thereto.

[0044] Unless otherwise specified, the methods described in the examples are conventional methods; the reagents used are commercially available unless otherwise specified.

[0045] Example 1:

[0046] A method for measuring the surface charge of biomaterials using a bubble probe includes the following steps:

[0047] (1) Optical Path Construction: An isosceles right-angled prism with a right-angled side length of 1mm and an aluminum-coated bevel (92% reflectivity) is connected to the bright surface of the fluid force support Holder using AB glue. After gluing, it is left to stand for 24 hours to fully fix the prism. A fluid force probe is placed on one side of the prism, with the isosceles right-angled prism approximately 1mm away from the probe, and its coated bevel facing the probe. A light source is placed on the other side of the fluid force probe, and the optical path reaches the bevel of the isosceles right-angled prism through the fluid force probe. During operation, a light source is provided on the side of the prism, and the light passes through the probe and is refracted by the prism into the objective lens, thus obtaining a side image of the bubble probe. The optical path is as follows: Figure 1 As shown, the optical path construction does not damage the Holder and does not affect the normal function of the Holder. It can obtain the side image of the bubble probe in real time, which is convenient for monitoring the bubble size and probe status.

[0048] (2) Probe Generation: A Micropipette fluid dynamic probe with a front opening diameter of 8 μm is installed on the fluid dynamic support holder in step (1) and connected to the fluid dynamic device. After applying a certain positive pressure, it is submerged in the liquid. The bubble at the tip can be observed through an isosceles right-angle prism using a side-provided light source. The probe is moved 5 μm at a time and photographed with a CCD. The actual size of the bubble can be obtained by calculating the ratio of the actual movement distance to the bubble size in the image. The bubble is stabilized at a suitable size by adjusting the pressure. After comparison, it was determined that the bubble is stable and of a suitable size at 250 mbar, which can produce a suitable bubble. The bubble probe obtained at 250 mbar in this embodiment is as follows: Figure 2 As shown, the bubble radius is approximately 4.1 μm, which allows it to remain stably on the probe tip for an extended period and can be used for further measurements. Excessive pressure will cause the bubble to become unstable and escape. A certain positive pressure must be applied to the probe before immersion in the liquid; otherwise, the liquid will fill the probe tip due to capillary action, preventing the formation of bubbles.

[0049] (3) Sample preparation: The biomaterial includes cells or phospholipid bilayers. If it is cells, proceed directly to step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid, DPPE phospholipid, or DPPG phospholipid in chloroform to form a lipid solution with a concentration of 1 mg·mL⁻¹. First, dispense the above solution into equal volumes in glass bottles and dry them overnight in a vacuum drying oven to form a lipid film. Then, add culture buffer to the bottle to resuspend the lipid film to a final concentration of 0.5 mg·mL⁻¹, thus obtaining a multilayer bilayer. The vesicle solution was sonicated in a water bath sonicator for 0.5 h at room temperature to obtain a small monolayer vesicle solution. Then, mica sheets with a diameter of 5 mm were cut using Scotch tape to obtain a fresh surface. 10 μL of vesicle solution was deposited onto the mica sheets to obtain lipid samples. The lipid samples were incubated above the phase transition temperature for 0.5 h and then cooled to room temperature. The DPTAP and DPPG samples were incubated at 60 °C, and the DPPE samples were incubated at 70 °C. The samples were then rinsed with imaging buffer. Finally, the prepared samples were stored in imaging buffer to prevent drying.

[0050] The culture buffer consisted of 10 ml HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 150 ml NaCl, and 5 ml CaCl2, with a pH of 7.3; the imaging buffer consisted of 10 ml HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), 150 ml NaCl, with a pH of 7.0.

[0051] In this embodiment, the obtained DPTAP phospholipid bilayer atomic force microscopy image is as follows: Figure 3 As shown in the figure, the obtained atomic force microscopy image of the DPPE phospholipid bilayer is as follows: Figure 4 As shown in the figure, the obtained atomic force microscopy image of the DPPG phospholipid bilayer is as follows: Figure 5 As shown in the figure, this embodiment successfully obtained a large area of ​​phospholipid bilayer on the mica surface.

[0052] (4) Sample Measurement: After the sample is prepared, it is fixed on the stage of the hydrodynamic microscope and measured in the imaging buffer environment. During the measurement, the pressure should be reduced during the needle insertion process. The pressure during the needle insertion process is 100 mbar. At this time, no bubbles are generated at the needle tip, so as to avoid contamination and disturbance of the bubbles during the needle insertion process. After the needle insertion is completed, the piezoelectric ceramic is raised to the highest point and restored to the normal pressure of 250 mbar. Force curve test is started. Force curve test is performed on three phospholipid samples, namely DPTAP, DPPE and DPPG, under the parameters of 10 nN pressure, 15 μm moving distance and 1 μm / s moving speed. The adhesion force between the bubble and the sample is obtained. 50 sets of data are collected for each sample.

[0053] By measuring the adhesion force, the magnitude of the electrostatic force between the bubbles and phospholipids is obtained, thus determining the surface charge of the sample. The adhesion force data between the three types of phospholipids and bubbles obtained in this embodiment are as follows: Figure 7 As shown in the figure, DPTAP phospholipids with a positive charge exhibit strong electrostatic interaction with the bubbles, with a relatively large adhesion force of 237.5 nN; neutral DPPE phospholipids have no electrostatic force with the bubbles, and the adhesion force is mainly capillary force, with a relatively small adhesion force of 4.3 nN; DPPG phospholipids with a negative charge exhibit strong electrostatic repulsion with the bubbles, with an adhesion force of 0 nN. The significant difference between the three indicates that the bubble probe can accurately detect the charge status on the surface of biomaterials.

[0054] The adhesion between phospholipids and air bubbles is composed of van der Waals forces, capillary forces, and electrostatic forces. Van der Waals forces are relatively small and have little impact. Capillary forces are mainly related to the surface tension of the solution and have essentially the same value in the same liquid environment. However, micro- and nano-sized air bubbles carry a weak negative charge in a typical aqueous environment. Therefore, the adhesion between phospholipids and air bubbles is mainly determined by the electrostatic force between them. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the air bubbles and phospholipids can be determined, thereby revealing the charge on the sample surface.

[0055] Capillary force is essentially a force primarily determined by the surface tension of the solution, with slight variations in the surface energy of different biomaterials. Therefore, this value is determined by the test solution environment. In this embodiment, the test environment is a 150 mmol saline solution (i.e., physiological saline concentration, with a small amount of buffer and Ca). 2+ Since different experimental conditions may vary, generally when the adhesion force is around 4-10 nN, the biomaterial is considered neutral and uncharged. Experimental data shows that when positively charged, electrostatic force dominates, while capillary force is actually very small (around 4.3 nN). Therefore, even for different samples, as long as the charge is consistent, the adhesion force values ​​will be relatively close; the only change is in the capillary force. Thus, values ​​above 230 nN can be considered positively charged.

[0056] Comparative Example 1

[0057] A method for measuring the surface charge of biomaterials using a bubble probe, as shown in Example 1, differs in that, during cultivation at the phase transition temperature, a culture buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.3) is not used; instead, a Ca-free buffer is used. 2+ Imaging buffer (10 mM HEPES, 150 mM NaCl, pH 7.0).

[0058] The other steps and conditions are the same as in Example 1.

[0059] The atomic force microscope image of the DPPG phospholipid bilayer prepared in this comparative example is as follows: Figure 6 As shown, no Ca was added during the process. 2+ Due to the loss of Ca 2+ The bridging effect prevents the formation of a large-area continuous phospholipid bilayer, resulting in multiple isolated island-like structures.

[0060] Comparative Example 2

[0061] A method for measuring the surface charge of biomaterials using a bubble probe, as shown in Example 1, except that the sample fixed on the sample stage is freshly peeled mica, rather than a biomaterial sample.

[0062] The other steps and conditions are the same as in Example 6.

[0063] The adhesion force data between mica and air bubbles obtained in this comparative example are as follows: Figure 8 As shown, since mica carries a negative charge in water, there is a strong electrostatic repulsion between it and the air bubble, and the adhesion force is 0 nN.

Claims

1. A method for measuring the surface charge of biomaterials using a bubble probe, characterized in that, The steps include the following: (1). Optical path construction: Connect the isosceles right-angled prism with aluminum-plated inclined surface to the bright surface of the fluid force support; set a fluid force probe on one side of the prism with the inclined surface of its plating facing the position of the probe, and set a light source on the other side of the fluid force probe. The optical path reaches the inclined surface of the isosceles right-angled prism through the fluid force probe. (2). Probe generation: The fluid force probe is installed on the fluid force support in step (1) and connected to the fluid force device. After applying a certain positive pressure, it is immersed in the liquid. The size of the tip bubble is calculated from the isosceles right-angle prism by the light source provided from the side. The bubble is stabilized by adjusting the pressure for further measurement. (3) Sample preparation: The biomaterial includes cells or phospholipid bilayers. If it is cells, proceed directly to step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid, DPPE phospholipid, or DPPG phospholipid in chloroform to form a lipid solution. First, dispense the above solution into equal amounts in glass bottles and dry them in a vacuum drying oven to form a lipid membrane. Then, add culture buffer to the bottle to resuspend the lipid membrane and obtain a multilayer vesicle solution. Place the solution in a water bath sonicator at room temperature to obtain a small monolayer vesicle solution. Then, cut the mica sheet with Scotch tape to obtain the surface and deposit the vesicle solution onto the mica sheet to obtain a lipid sample. After culturing the lipid sample above the phase transition temperature, cool it to room temperature and rinse it with imaging buffer. Finally, use imaging buffer to preserve the prepared sample. (4). Sample measurement: After the sample is prepared, it is fixed on the sample stage of the hydrodynamic microscope and measured in the imaging buffer environment. During the measurement, no air bubbles are generated at the tip of the needle during the needle insertion process. After the needle insertion is completed, the piezoelectric ceramic is raised to the highest point and restored to normal pressure. Force curve test is started to obtain the adhesion force between the air bubble and different samples. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the air bubble and phospholipid is obtained, and then the charge status of the sample surface is obtained. When the adhesion force is 230 nN or above, the biomaterial is judged to be positively charged. When the adhesion force is 4 nN-10 nN or below, the biomaterial is judged to be neutral and uncharged. When the adhesion force is 0 nN, the biomaterial is judged to be negatively charged.

2. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, Step (1) includes one or more of the following schemes: a. The length of the right-angled side of the isosceles right-angled prism is 1mm; b. The reflectivity of the aluminum-coated inclined surface of the isosceles right-angle prism is 92%; c. The connection between the fluid force support and the isosceles right-angle prism is to use AB glue for bonding. After applying the glue, let it stand for 24 hours to fix the prism. d. The isosceles right-angle prism is 1 mm away from the probe position.

3. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, In step (2), the diameter of the opening at the tip of the hydrodynamic probe is 8 μm.

4. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, In step (2), the pressure is adjusted to 250 mbar, at which point the bubble radius is 4.1 μm.

5. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, In step (2), the method for calculating the bubble radius is to move the probe 5 μm each time in the liquid, and calculate the actual size of the bubble by the ratio of the actual moving distance to the bubble size in the image.

6. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, Step (3) includes one or more of the following schemes: I. The lipid solution concentration formed is 1 mg·mL⁻¹, and the lipid membrane is resuspended to a final concentration of 0.5 mg·mL⁻¹; II. The ultrasonic time for the water bath ultrasonic instrument is 0.5 hours; III. The mica sheet has a diameter of 1 cm, and the vesicle solution deposited on the mica sheet is 10 μL; IV. The DPTAP and DPPG samples were incubated at 60℃, and the DPPE samples were incubated at 70℃ for 0.5 h. V. The culture buffer consists of 10 ml of 4-hydroxyethylpiperazine ethanesulfonic acid, 150 ml of NaCl, and 5 ml of CaCl2, with a pH of 7.

3. VI. The imaging buffer consisted of 10 ml of 4-hydroxyethylpiperazine ethanesulfonic acid and 150 ml of NaCl, with a pH of 7.

0.

7. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, In step (4), the pressure during the needle insertion process is 100 mbar, at which point no bubbles are generated at the needle tip; after the needle insertion is completed, the piezoelectric ceramic is raised to its highest position, and the pressure is restored to 250 mbar.

8. The method for measuring the surface charge of biomaterials using a bubble probe according to claim 1, characterized in that, In step (4), during the force curve test, the pressure is set to 10 nN, the moving distance is 15 μm, and the moving speed is 1 μm / s.

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