Method for measuring surface charge of biological material by using bubble probe

Abstract

The invention provides a method for measuring surface charges of a biological material by using a bubble probe, and belongs to the technical field of physical and chemical instrument analysis. Comprising the steps that an isosceles right-angle prism with a back coating is pasted on a fluid force Holder through AB glue, then a fluid force probe is assembled, the fluid force probe is immersed in water after a certain positive pressure is applied, real-time observation and size measurement of bubbles are achieved by providing a lateral light source, then force curve measurement is conducted under set parameters through software, and the measurement result is obtained. The charged condition of the biological material can be judged through the adhesive force of the bubbles and the biological material. Through fluid force equipment and a lateral light path system, stable micron-sized bubbles with controllable size are generated at the front end of the probe, the condition that the surface of a biological material is charged can be measured by utilizing the bubbles, and the device has the advantages of rapidness, simplicity, convenience, in-situ performance and small damage.

Description

Technical Field

[0001] The present invention relates to a method for measuring the surface charge of biological materials using a bubble probe, belonging to the technical field of physical and chemical instrumental analysis. Background Art

[0002] Surface charge widely exists on the surface of biological materials and is closely related to many dynamic equilibria and physiological processes. Its sign and density largely determine the properties of the surface. For example, the surface charge of cells is mainly determined by the charge of the lipid bilayer and proteins of the cell membrane, which may vary depending on the species, cell type, and differentiation state. It has been widely confirmed that cell surface charge affects many membrane-regulated cell functions, such as endocytosis, muscle cell 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. There have been reports on the changes in the cell surface charge of various types of stem cells during the differentiation process, as well as the differences between cancer cells and normal cells. Therefore, the ability to identify the surface charge of different types of living cells and the ability to visualize the local surface charge of cells are of great significance for understanding the basic functions of many cells. However, due to the lack of a robust technology capable of performing micro-nano scale measurements in complex environments, the measurement and mapping of the surface charge of biological materials remain a major challenge. Traditional techniques such as Zeta potential measurement or potentiometric titration cannot provide information on the charge distribution at the interface of biological materials, nor can they measure the charge of tiny materials such as single cells. Recently, scanning ion conductance microscopy has been used for mapping the surface charge of biological materials. This method is based on the principle of ion current rectification and uses a single nano-pipette to measure the sample topography and surface charge density. However, the nano-pipette needs to first approach the edge of the sample and then return to the neutral position by trial and error, which is a cumbersome process and requires a very long time to scan the sample surface point by point. Therefore, this technique cannot perform rapid surface charge characterization.

[0003] The fluid force microscope is an atomic force microscope (AFM) based on a hollow cantilever, which can achieve liquid dispensing and single living cell stimulation under physiological conditions. Since its birth in 2009, the fluid force microscope has become the most promising tool for biological research at the micro-nano scale. The fluid force microscope combines the precise force control of AFM with the versatility of microfluidics, integrates micro-sized channels in the AFM cantilever, and connects to a vacuum pump through an injection hose to form a continuous and closed channel. This channel can be filled with any selected solution and can be immersed in a liquid environment. There is a small hole at the AFM tip at the end of the cantilever, which can be used for liquid dispensing and cell manipulation, and is mostly used for pipetting, and there is no relevant report on charge measurement. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for generating stable bubbles at the tip of a probe using a fluid force microscope, thereby realizing the measurement of the surface charge of different biological materials. In a general liquid environment, the surface of the bubble will carry a weak negative charge. The magnitude of the adhesion force between the bubble and different biological materials 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 judged, and then the charge condition of the sample surface can be obtained. The bubbles generated by the fluid force microscope probe of the present invention are stable and controllable in size, much smaller than the bubbles of ordinary probes, can stably exist at the tip of the needle and are not easy to break away, and will not damage the sample surface, which is very suitable for the detection of the surface of biological samples. Using this probe in combination with an atomic force microscope to detect the surface charge of biological materials, the applicable samples are wide, the scanning process is simple to operate, the scanning time required is short, the differences between the detection results of different charges are large, and the detection accuracy is high, which can give full play to the advantages of the bubble probe and has broad application prospects in the fields of physical chemistry, biology, medicine, etc.

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

[0006] A method for measuring the surface charge of biological materials using a bubble probe, comprising the following steps:

[0007] (1). Optical path construction: Connect an isosceles right prism with an aluminized inclined surface to the bright surface of the fluid force holder Holder using AB glue; a fluid force probe is arranged on one side of the prism, and its plated inclined surface should face the position of the probe. A light source is arranged on the other side of the fluid force probe, and the optical path reaches the inclined surface of the isosceles right 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 into the objective lens by the prism, so that a side image of the bubble probe can be obtained.

[0008] (2). Probe generation: Install the fluid force probe on the fluid force holder 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 of the needle can be calculated from the isosceles right prism through the laterally provided light source. Adjust the pressure to make the bubble stable at an appropriate size for further measurement; a certain positive pressure needs to be applied to the probe before immersing it in the liquid, otherwise the liquid will fill the tip of the probe due to capillary action and no bubbles can be generated;

[0009] Preferably according to the present invention, step (1) includes any one or more of the following schemes:

[0010] a. The right-angled side length of the isosceles right prism should be 1 mm. A too small prism is difficult to operate and more expensive, and a too large prism will cause the optical path to become longer and the objective lens cannot focus on the tip of the probe;

[0011] b. The reflectivity of the aluminized inclined surface of the isosceles right prism is 92%;

[0012] c. The connection method between the hydrodynamic holder and the isosceles right prism is to use AB glue for bonding. After applying the glue and bonding, let it stand for 24 h to fully fix the prism.

[0013] d. The isosceles right prism is about 1 mm away from the probe position. Too far a distance will cause the optical path to become longer, making it impossible for the objective lens to focus on the probe tip.

[0014] According to the preference of the present invention, in step (2), the front-end opening diameter of the hydrodynamic probe is a Micropipette probe with a diameter of 8 μm.

[0015] According to the preference of the present invention, in step (2), adjust the pressure to 250 mbar to generate appropriate bubbles. At this time, the bubble radius is about 4.1 μm. Excessive pressure will cause the bubbles to be unstable and escape.

[0016] According to the preference 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 through the ratio of the actual moving distance to the bubble size in the image.

[0017] (3) Sample preparation: The biological material includes cells or phospholipid bilayers. If it is cells, directly enter step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid or DPPE phospholipid or DPPG phospholipid in chloroform to form a lipid solution; first, equally divide the above solution into glass bottles and place them in a vacuum drying oven to dry overnight to form a lipid film; then add culture buffer to the bottles to resuspend the lipid film to obtain a multi-lamellar vesicle solution, and place it in a water bath sonicator at room temperature to sonicate to obtain a small unilamellar vesicle solution; then, cut a mica sheet with Scotch tape to obtain a fresh surface, deposit the vesicle solution on the mica sheet to obtain a lipid sample, culture the lipid sample above the phase transition temperature and then cool it to room temperature, and rinse it with imaging buffer; finally, use imaging buffer to store the prepared sample to prevent drying.

[0018] (4) Sample measurement: After the sample is prepared, fix it on the hydrodynamic microscope sample stage and perform measurements in an imaging buffer environment; during measurement, maintain no bubbles at the tip of the needle during the needle-lowering process. After the needle-lowering is completed, lift the piezoelectric ceramic to the highest position, restore to the normal pressure, and start the force curve test to obtain the adhesion force between the bubble and different phospholipid samples; by measuring the magnitude of the adhesion force, obtain the magnitude of the electrostatic force between the bubble and the phospholipid, and then obtain the charged situation on the sample surface; when the adhesion force is 230 nN or more, it is judged that the biological material is positively charged. When the adhesion force is between 4 nN and 10 nN, it is judged that the biological material is neutral and uncharged. When the adhesion force is 0 nN, it is judged that the biological material is negatively charged.

[0019] The adhesion force between phospholipids and bubbles consists of van der Waals force, capillary force and electrostatic force. The van der Waals force is relatively small and has little effect. The capillary force is mainly related to the surface tension of the solution and has basically the same value in the same liquid environment. Micro-nano bubbles carry a weak negative charge in a general water environment. Therefore, the adhesion force between phospholipids and 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 bubbles and phospholipids can be judged, and then the charge situation on the sample surface can be obtained.

[0020] Preferably according to the present invention, step (3) includes one or more of the following any schemes:

[0021] Ⅰ. The concentration of the formed lipid solution 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 of the water bath ultrasonic instrument is 0.5 h;

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

[0024] Ⅳ. The culture temperature of the DPTAP sample and the DPPG sample is 60 °C, and the culture temperature of the DPPE sample is 70 °C, and the culture time is 0.5 h for both;

[0025] Ⅴ. The culture buffer composition is 10 ml of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 150 ml of NaCl, 5 ml of CaCl 2 , and the pH of the solution is 7.3;

[0026] Ⅵ. The imaging buffer composition is 10 ml of HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 150 ml of NaCl, and the pH of the solution is 7.0.

[0027] Preferably according to 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, and there are no bubbles generated at the tip of the needle at this time, 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 lifted to the highest, and the pressure is restored to 250 mbar.

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

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

[0030] 1. The present invention provides a new method for manufacturing a bubble probe. In the traditional bubble probe, a hydrophobized gold sheet is first pasted at the tip of a cantilever without a needle tip. Then, air bubbles are carefully injected into the liquid pool through a super-tip pipette. Subsequently, the cantilever is slowly lowered to pick up a suitable bubble using hydrophobic interaction. However, the bubbles obtained by this method are often relatively large, with a radius of about 100 μm. When measuring relatively small samples, they are easily interfered by the substrate. Moreover, when measuring samples with a relatively large adhesion force, the bubbles are likely to fall off from the probe tip, and accurate adhesion force cannot be obtained. On the other hand, since gas slowly dissolves in water, the size of its bubbles is not constant and will decrease over time, resulting in deviation of experimental results. The bubble probe obtained by the present invention using fluid force microscopy technology has a stable and controllable bubble size, and the size is much smaller than that of the bubbles of the previous bubble probes. It can stably exist at the needle tip and is not easy to detach, and will not cause damage to the sample surface, which is very suitable for the detection of the surface of biological samples.

[0031] 2. The present invention provides a method for constructing an optical path for observing the tip of a fluid force probe from the side. As an instrument with great prospects in the biological field, a fluid force microscope is generally equipped with an inverted microscope and a top microscope, which can observe the upper and lower surfaces of the probe, but cannot observe the side of the probe and cannot well analyze the real-time state. We provide a set of lateral optical path systems. The light source provided externally is refracted by a prism and enters the objective lens, enabling the real-time observation of the probe state from the side. The present invention realizes the real-time observation and size measurement of the needle tip bubbles through this lateral optical path system, ensuring the accuracy of experimental results.

[0032] 3. The present invention prepares a large-area phospholipid bilayer with different charges on a mica substrate. First, phospholipid multilamellar vesicles are formed, and then small unilamellar vesicles are obtained by ultrasonic method. Heating causes the vesicles to rupture and spread on mica to form a stable phospholipid bilayer. Different from the phospholipids obtained by traditional methods, which are generally in small areas, the present invention obtains a whole piece of phospholipid uniformly covering the mica surface by regulating the phospholipid solution concentration, incubation temperature, ultrasonic time, and Ca 2+ concentration. The area is large and it is applicable to phospholipids with different charge types, and can simulate the charge conditions of various biological material surfaces.

[0033] 4. The present invention provides a method for measuring the surface charge of biological materials using a bubble probe. Taking the phospholipid bilayer as an example to simulate the surface of biological materials under different charge conditions, in a general liquid environment, the surface of the bubble will carry a weak negative charge. The magnitude of the adhesion force between the bubble and different biological materials 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 judged, and then the charge situation on the surface of the sample can be obtained. Through experimental tests, it is found that the adhesion force between phospholipids with different charges and the bubble is significantly different, and it has the advantages of a wide range of applied samples, simple operation during the scanning process, short scanning time, and high detection accuracy. Description of the Drawings

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

[0035] Wherein: 1, light source; 2, fluid force bracket; 3, isosceles right prism; 4, sample; 5, objective lens; 6, probe;

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

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

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

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

[0040] Figure 6 is the phospholipid bilayer isolated island structure diagram without adding Ca 2+ formed;

[0041] Figure 7 is a box plot of the adhesion force data between three different charged phospholipids and the bubble;

[0042] Figure 8 is a box plot of the adhesion force data between mica and the bubble and the adhesion force data between phospholipids and the bubble. Detailed Embodiments

[0043] The present invention will be further described below by way of examples in conjunction with the drawings, but not limited thereto.

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

[0045] Example 1:

[0046] A method for measuring the surface charge of biological materials using a bubble probe, comprising the following steps:

[0047] (1). Optical path construction: An isosceles right prism with a right-angled side length of 1 mm and an aluminized inclined surface (reflectivity of 92%) is connected to the bright surface of the fluid force holder Holder using AB glue. After gluing, it is left standing for 24 h to fully fix the prism; a fluid force probe is arranged on one side of the prism, about 1 mm away from the probe position of the isosceles right prism, and its plated inclined surface should face the position of the probe. A light source is arranged on the other side of the fluid force probe, and the optical path reaches the inclined surface of the isosceles right 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 into the objective lens by the prism, so that a side image of the bubble probe can be obtained. The optical path is as Figure 1 shown. It can be seen that the optical path construction does not damage the Holder and does not affect the normal function of the Holder. The side image of the bubble probe can be obtained in real time, which is convenient for monitoring the bubble size and the probe state.

[0048] (2). Probe generation: A Micropipette fluid force probe with a front-end opening diameter of 8 μm is installed on the fluid force holder Holder in step (1) and connected to the fluid force device. After applying a certain positive pressure, it is immersed in the liquid. After observing the bubble at the tip from the isosceles right prism through the laterally provided light source, the probe is moved 5 μm each time and a photo is taken with a CCD. By calculating the ratio of the actual moving distance to the bubble size in the image, the actual size of the bubble can be obtained. By adjusting the pressure, the bubble is stabilized at a suitable size. After comparison, it is determined that the bubble is stable and has a suitable size at 250 mbar and can generate a suitable bubble. The bubble probe obtained at 250 mbar in this embodiment is as Figure 2 shown. At this time, the bubble radius is about 4.1 μm, and it can stably exist on the tip for a long time and can be used for further measurement. Excessive pressure will cause the bubble to be unstable and escape. A certain positive pressure needs to be applied to the probe before immersing it in the liquid, otherwise the liquid will fill the tip of the probe due to capillary action and no bubble can be generated.

[0049] (3). Sample preparation: The biological material includes cells or phospholipid bilayers. If it is cells, directly proceed to step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid or DPPE phospholipid or DPPG phospholipid in chloroform to form a lipid solution with a concentration of 1 mg·mL-1. First, equally divide the above solution into glass bottles and place them in a vacuum drying oven to dry overnight to form a lipid film. Then, add the culture buffer to the bottles to resuspend the lipid film to a final concentration of 0.5 mg·mL-1 to obtain a multilamellar vesicle solution. Place it in a water bath sonicator at room temperature and sonicate for 0.5 h to obtain a small unilamellar vesicle solution. Then, cut a mica sheet with a diameter of 5 mm with Scotch tape to obtain a fresh surface, deposit 10 μL of the vesicle solution onto the mica sheet to obtain a lipid sample. Incubate the lipid sample at a temperature above the phase transition temperature for 0.5 h and then cool it to room temperature. The incubation temperature for DPTAP samples and DPPG samples is 60 °C, and the incubation temperature for DPPE samples is 70 °C. Rinse with the imaging buffer. Finally, use the imaging buffer to store the prepared sample to prevent drying.

[0050] The culture buffer consists of 10 ml of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 ml of NaCl, and 5 ml of CaCl 2 , and the pH of the solution is 7.3; the imaging buffer consists of 10 ml of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 ml of NaCl, and the pH of the solution is 7.0.

[0051] In this example, the atomic force microscope images of the DPTAP phospholipid bilayer are as shown in Figure 3 shown, and the atomic force microscope images of the DPPE phospholipid bilayer are as shown in Figure 4 shown, and the atomic force microscope images of the DPPG phospholipid bilayer are as shown in Figure 5 shown. Thus, it can be seen that a large-area phospholipid bilayer was successfully obtained on the mica surface in this example.

[0052] (4). Sample measurement: After the sample is prepared, fix it on the fluid force microscope sample stage and perform measurements in an imaging buffer environment. During measurement, the pressure should be reduced during the needle insertion process. The pressure during the needle insertion process is 100 mbar, and there are no bubbles generated at the tip at this time, thus avoiding contamination and disturbance of the bubbles during the needle insertion process. After the needle insertion is completed, lift the piezoelectric ceramic to the highest position and restore it to the normal pressure of 250 mbar. Start the force curve test. Perform force curve tests on the three phospholipid samples of DPTAP, DPPE, and DPPG under the parameters of a set pressure of 10 nN, a moving distance of 15 μm, and a moving speed of 1 μm / s, respectively, to obtain the adhesion force between the bubble and the sample. Statistically analyze 50 groups of data for each sample.

[0053] By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the bubble and the phospholipid is obtained, and then the charge situation on the surface of the sample is derived. The adhesion force data of the three phospholipids and the bubble obtained in this embodiment are as Figure 7 shown. It can be seen that there is a strong electrostatic interaction between the DPTAP phospholipid with one positive charge and the bubble, and the adhesion force is relatively large, which is 237.5 nN; there is no electrostatic force between the neutral DPPE phospholipid and the bubble, and the adhesion force is mainly capillary force, which is relatively small, 4.3 nN; there is a strong electrostatic repulsion between the DPPG phospholipid with one negative charge and the bubble, and the adhesion force is 0 nN. The difference among the three is large, indicating that the bubble probe can accurately detect the charge situation on the surface of biological materials.

[0054] The adhesion force between the phospholipid and the bubble is composed of van der Waals force, capillary force and electrostatic force. The van der Waals force is relatively small and has little influence. The capillary force is mainly related to the surface tension of the solution and is basically the same in the same liquid environment. And micro-nano bubbles carry a weak negative charge in a general water environment. Therefore, the adhesion force between the phospholipid and the bubble is mainly determined by the electrostatic force between the two. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the bubble and the phospholipid can be judged, and then the charge situation on the surface of the sample can be derived.

[0055] The capillary force is essentially a force mainly determined by the surface tension of the solution, and the surface energy of different biological materials also has a little influence. Therefore, this value will be determined by the test solution environment. The test environment in this embodiment is 150 mmol salt solution (i.e., the concentration of physiological saline, and there is also a little buffer and Ca 2+ ).). Due to possible fluctuations under different experimental conditions, generally when the adhesion force is about 4 - 10 nN, it is judged that the biological material is neutral and uncharged. From the experimental data, when it is positively charged, the electrostatic force is dominant and the capillary force is actually very small (about 4.3 nN). Therefore, even for different samples, as long as the charge situation is the same, the value of the adhesion force will be relatively close, and only a little change in the capillary force occurs. Therefore, it can be considered positively charged above 230 nN.

[0056] Comparative Example 1

[0057] A method for measuring the surface charge of biological materials using a bubble probe is as shown in Example 1. The difference is that when culturing at the phase transition temperature, the culture buffer (10 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , pH 7.3) is not used, but an imaging buffer without Ca 2+ (10 mM HEPES, 150 mM NaCl, pH 7.0) is used.

[0058] Other steps and conditions are the same as those in Example 1.

[0059] The atomic force microscope image of the DPPG phospholipid bilayer prepared in this comparative example is as shown in Figure 6 , and no Ca was added in this process. 2+ , due to the loss of the bridging effect of Ca 2+ , a large-area continuous phospholipid bilayer could not be formed, but multiple isolated island-like structures were formed.

[0060] Comparative Example 2

[0061] A method for measuring the surface charge of a biological material using a bubble probe, as shown in Example 1, except that the sample fixed on the sample stage is freshly exfoliated mica instead of a biological material sample.

[0062] Other steps and conditions are the same as those in Example 6.

[0063] The adhesion force data of mica and bubbles obtained in this comparative example are as shown in Figure 8 . Since mica is negatively charged in water, there is a strong electrostatic repulsion between mica and bubbles, and the adhesion force is 0 nN.

Claims

1. A method for measuring the surface charge of a biomaterial using a bubble probe, characterized in that: The steps include: (1) Optical path construction: connect an isosceles right-angle prism with aluminum-plated bevel to the bright surface of the fluid force bracket; set a fluid force probe on one side of the prism with its beveled surface facing the probe, and set a light source on the other side of the fluid force probe. The optical path passes through the fluid force probe to the bevel of the isosceles right-angle prism; (2) Probe generation: The fluid force probe is mounted on the fluid force bracket 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 bubble at the needle tip is calculated from the isosceles right-angle prism through the light source provided from the side. The bubble is stabilized by adjusting the pressure for further measurement. (3) Sample preparation: The biological material includes cells or phospholipid bilayers. If it is a cell, directly proceed to step (4). If it is a phospholipid bilayer, dissolve powdered DPTAP phospholipid or DPPE phospholipid or DPPG phospholipid in chloroform to form a lipid solution; first, divide the above solution into glass bottles in equal amounts, place them in a vacuum drying oven to dry, and form a lipid film; then add culture buffer to the bottle to resuspend the lipid film to obtain a multilayer vesicle solution, and then place it in a water bath sonicator for sonication at room temperature to obtain a small unilamellar vesicle solution; then, cut the mica sheet with Scotch tape to obtain a surface, and deposit the vesicle solution on the mica sheet to obtain a lipid sample. After the lipid sample is cultured above the phase transition temperature, it is cooled to room temperature and rinsed with imaging buffer; finally, the prepared sample is preserved with imaging buffer; (4) Sample measurement: After the sample is prepared, it is fixed on the sample stage of the fluid force microscope and measured in an imaging buffer environment. During the measurement, the needle tip is kept free of bubbles during the needle insertion process. After the needle insertion is completed, the piezoelectric ceramic is lifted to the highest position and restored to normal pressure. The force curve test is started to obtain the adhesion force between the bubble and different samples. By measuring the magnitude of the adhesion force, the magnitude of the electrostatic force between the bubble and the phospholipid is obtained, and then the charge of the sample surface is obtained. When the adhesion force is 230nN or above, the biomaterial is judged to be positively charged. When the adhesion force is 4nN-10nN or below, the biomaterial is judged to be neutral and uncharged. When the adhesion force is 0nN, the biomaterial is judged to be negatively charged.

2. The method for measuring the surface charge of biological materials using a bubble probe according to claim 1, characterized in that: Step (1) includes one or more of the following: a. The length of the right angle side of the isosceles right angle prism is 1 mm; b. The reflectivity of the aluminum-plated bevel of the isosceles right-angle prism is 92%; c. The connection between the fluid force bracket and the isosceles right-angle prism is to use AB glue for bonding. After the glue is applied and bonded, the prism is left to stand for 24 hours to fix it. d. The isosceles right prism is 1 mm away from the probe.

3. The method for measuring the surface charge of biological materials using a bubble probe according to claim 1, characterized in that: In step (2), the diameter of the front opening of the fluid force probe is 8 μm.

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

5. The method for measuring the surface charge of biological materials 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 in the liquid by 5 μm each time, and calculate the actual size of the bubble by the ratio of the actual moving distance in the image to the bubble size.

6. The method for measuring the surface charge of biological materials using a bubble probe according to claim 1, characterized in that: Step (3) includes one or more of the following: Ⅰ, the concentration of the formed lipid solution was 1 mg·mL-1, and the lipid film was resuspended to a final concentration of 0.5 mg·mL-1; II, water bath sonication time was 0.5 h; III, the diameter of the mica sheet is 1 cm, and the vesicle solution deposited on the mica sheet is 10 μL; IV, the incubation temperature of DPTAP and DPPG samples was 60°C, the incubation temperature of DPPE samples was 70°C, and the incubation time was 0.5 h; V, the composition of the culture buffer is 10 ml 4-hydroxyethylpiperazineethanesulfonic acid, 150 ml NaCl, 5 ml CaCl2, and the solution pH is 7.3; VI, imaging buffer composition is 10 ml 4-hydroxyethylpiperazineethanesulfonic acid, 150 ml NaCl, solution pH is 7.

0.

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

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

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