A method for quantitatively measuring adhesion between bacteria and cells based on fluid force microscopy

Abstract

The present disclosure provides a method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy, comprising: culturing bacteria and cells separately, adding bacteria to the cells to allow the bacteria and cells to interact; selecting a fluid force microscopy probe and pre-treating the probe; searching for a single live bacterium to be captured in a liquid environment where the bacteria and cells interact, applying negative pressure using a fluid force microscopy pressure controller to cause the pinhole at the probe tip to absorb the bacteria, thereby obtaining a fluid force microscopy probe that captures a single bacterium; bringing the fluid force microscopy probe that captures a single bacterium close to the cell surface at a preset speed, staying there for a preset time when a preset force is reached, and then returning to the probe to obtain adhesion force curve data between the bacteria and cells. Utilizing the present disclosure, the degree of interaction between a single bacterium and a cell can be accurately characterized, and the adhesion force between a single bacterium and a cell can be efficiently and quantitatively measured.

Description

Technical Field

[0001] The present disclosure relates to the technical field of bacterial adhesion characterization, and in particular to a method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy. Background Art

[0002] Due to the overuse of antibiotics, bacterial infections, particularly those caused by antibiotic-resistant bacteria, have become one of the most common infections worldwide, leading to numerous diseases and seriously endangering human life. Bacterial-cell adhesion directly reflects the degree of interaction between the two and is a key factor in bacterial pathogenicity. Previous studies have primarily characterized the adhesion between bacterial adhesins and specific receptors at the single-molecule scale. However, quantitative measurement of the adhesion of individual bacteria remains a significant challenge.

[0003] Currently, techniques such as atomic force microscopy provide quantitative measurements of the adhesion forces between bacteria and biomaterial surfaces. However, when using AFM, the bacteria are irreversibly fixed to the probe cantilever to create a modified probe. This makes it impossible to replace the modified probe during the measurement process, resulting in limited measurement throughput and inaccurate subsequent adhesion force measurements. Furthermore, due to the limited measurement range, AFM quantitative measurement methods are not suitable for measuring the adhesion forces between bacteria and cells.

[0004] In view of this, it is necessary to provide a new method to accurately and rapidly measure the adhesion force between single bacteria and cells. Summary of the Invention

[0005] (1) Technical issues to be solved

[0006] In response to technical problems such as the difficulty in replacing bacterial modified probes during the measurement of bacterial adhesion force in traditional methods, the present disclosure provides a method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy, so as to achieve reversible capture of bacteria and quantitative measurement of the adhesion force between single bacteria and cells.

[0007] (2) Technical solution

[0008] To achieve the above objectives, the present disclosure provides a method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy, comprising:

[0009] Cultivate bacteria and cells separately, add bacteria to the cells, and allow the bacteria and cells to interact;

[0010] Select a fluid force microscope probe and pre-treat the probe;

[0011] Searching for a single live bacterium to be captured in a liquid environment where bacteria and cells interact, using a fluid force microscope pressure controller to apply negative pressure so that the pinhole at the probe tip absorbs the bacterium, thereby obtaining a fluid force microscope probe that captures a single bacterium; and

[0012] The fluid force microscope probe that captures a single bacterium is brought close to the cell surface at a preset speed. When the preset force is reached, it stays for a preset time and then returns to the probe to obtain the adhesion force curve data between the bacteria and the cell.

[0013] In the above scheme, in the steps of culturing bacteria and cells separately, the bacteria are cultured by culturing Gram-positive bacteria or Gram-negative bacteria under the combined action of different physical factors and different chemical factors, wherein: the physical factor is at least one of bacterial density and fluid shear force; the chemical factor is at least one of reagent type and reagent concentration; the Gram-positive bacteria are at least one of Staphylococcus, Streptococcus and Lactobacillus; and the Gram-negative bacteria are at least one of Escherichia coli, Pseudomonas aeruginosa and Shigella.

[0014] In the above scheme, in the steps of culturing bacteria and cells separately, the cultured cells are cultured adherent cells under the combined action of different physical factors and different chemical factors, wherein: the physical factors are at least one of cell density, extracellular matrix stiffness and geometric constraints of cells; and the chemical factors are at least one of reagent type and reagent concentration.

[0015] In the above scheme, in the step of adding bacteria to the cells to allow the bacteria to interact with the cells, the cells are cultured in a phenol red-free culture medium containing 10% fetal bovine serum that has been filtered through a 0.22 μm filter membrane under treatment with different chemical factors, wherein the chemical factor is at least one of the type of reagent and the concentration of the reagent, and the filtration is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe; bacteria are added to the cells and allowed to interact with the cells at a temperature of 37°C and a gas environment of 5% CO2 and 95% O2.

[0016] In the above solution, the step of selecting a fluid force microscope probe includes: selecting a fluid force microscope probe with a pinhole diameter of 300nm-1000nm and a spring constant of 0.6-2N / m.

[0017] In the above scheme, the step of pre-treating the probe includes: adding sample liquid to the probe sample pool, modifying the probe, and semi-automatically measuring the spring constant k and sensitivity S of the fluid force microscope probe for subsequent calculation of the adhesion force.

[0018] In the above scheme, the sample liquid is a mixed solution of 10X trypsin and fluorescent dye that has been subjected to high-speed centrifugation at a speed of 14,800 rpm for 5-20 minutes, wherein the volume ratio of 10X trypsin to fluorescent dye is 3:1; high-speed centrifugation of the sample liquid is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe, the trypsin in the sample liquid is used to prevent cells from adhering to the probe during the measurement process, and the fluorescent dye is used to determine whether the state of the probe pinhole is unobstructed; the modification liquid is 0.1 mg / ml poly(L-lysine)-grafted-poly(ethylene glycol) that has been subjected to high-speed centrifugation at a speed of 14,800 rpm for 5-20 minutes, and the immersion time is greater than 20 minutes; high-speed centrifugation of the modification liquid is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe, and the modification liquid is used to prevent bacteria from irreversibly adhering to the fluid force microscope probe.

[0019] In the above scheme, in the step of using the fluid force microscope pressure controller to apply negative pressure to make the pinhole at the tip of the probe absorb bacteria, the negative pressure applied by the pressure controller is -50 to -800 mbar, and the negative pressure enables the pinhole at the tip of the fluid force microscope probe to capture bacteria.

[0020] In the above scheme, the fluid force microscope probe that captures a single bacterium approaches the cell surface at a preset speed, stays for a preset time when the preset force is reached, and then returns to the probe to obtain the adhesion force curve data between the bacteria and the cell. In this step, the preset speed is 0.1-5 μm / s, the preset force is 10-100 nN, the preset time is 1-300 s, and the return distance is 20-50 nm.

[0021] In the above scheme, after obtaining the adhesion force curve data between bacteria and cells, the method further includes: calculating the adhesion force between bacteria and cells using an adhesion force calculation formula based on the obtained adhesion force curve data between bacteria and cells, wherein the adhesion force calculation formula is:

[0022] F=USk

[0023] Where U(v) is the voltage difference from when the probe is subjected to the adhesion force to when it returns to the initial state, S(m / v) is the probe sensitivity, and k(N / m) is the spring constant.

[0024] (3) Beneficial effects

[0025] The method provided in the present disclosure for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy has the following advantages over existing technologies:

[0026] 1. The method provided by the present disclosure for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy achieves reversible capture of bacteria, efficient and quantitative measurement of the adhesion force between individual bacteria and cells, and can accurately characterize the degree of interaction between individual bacteria and cells.

[0027] 2. The method provided by the present disclosure for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy can achieve rapid and reversible capture of single bacteria, which is conducive to the replacement of bacteria after each measurement, and overcomes the shortcomings of traditional adhesion force measurement, such as the difficulty in replacing bacteria-modified probes during measurement.

[0028] 3. The method provided in the present disclosure for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy can not only realize the measurement of the adhesion force between traditional bacteria and the surface of biomaterials, but can also be applied to the quantitative measurement of the adhesion force between individual bacteria and cells under the action of different physical and chemical factors.

[0029] 4. The method provided in the present disclosure for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy has simple operation, fast and efficient measurement process, and accurate measurement results. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more fully understand the present disclosure and its advantages, a detailed description of the accompanying drawings is now given, in which:

[0031] Figure 1 4 is a flow chart of a method for quantitatively measuring adhesion force between bacteria and cells based on fluid force microscopy according to an embodiment of the present disclosure.

[0032] Figure 2 Schematic diagram of measuring the adhesion force between bacteria and cells based on fluid force microscopy in Example 1.

[0033] Figure 3 1 is a graph showing the adhesion force curve between Staphylococcus aureus and IEC-6 cells in Example 1.

[0034] Figure 4 This is a graph showing the adhesion force curve between Escherichia coli and IEC-6 cells in Example 2.

[0035] Figure 5 3 is a graph showing the adhesion force data between Staphylococcus aureus and IEC-6 cells at different contact times using the fluid force microscope probe in Example 3.

[0036] Figure 6 3 is a graph showing the adhesion force data between Staphylococcus aureus and IEC-6 cells at different moving speeds of the fluid force microscope probe in Example 4.

[0037] Figure 7This is a graph showing the adhesion force between Staphylococcus aureus and IEC-6 cells measured by fluid force microscopy under the regulation of different extracellular matrix stiffnesses in Example 5. DETAILED DESCRIPTION

[0038] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present disclosure. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0039] Fluid force microscopy (FliudFM) is a latest-generation single-cell manipulation system that combines atomic force microscopy and microfluidics. Because the FFM probe cantilever is hollow, a pressure controller can apply positive or negative pressure within the cantilever channel to control the flow of fluid, reversibly capturing individual bacteria at the pinhole at the probe tip. This system, equipped with high-precision control equipment and highly automated operation, enables rapid and accurate quantitative measurement of bacterial-cell adhesion forces.

[0040] The present disclosure provides a method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy. Figure 1 As shown, Figure 1 This is a flow chart of a method for quantitatively measuring the adhesion force between bacteria and cells based on a fluid force microscope according to an embodiment of the present disclosure. It should be noted that Figure 1 The examples shown are merely examples of application scenarios in which the embodiments of the present disclosure can be applied, to help those skilled in the art understand the technical content of the present disclosure, but do not mean that the embodiments of the present disclosure cannot be used in other environments or scenarios.

[0041] like Figure 1 As shown, the method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy in an embodiment of the present disclosure includes the following steps:

[0042] Step S1: Cultivating bacteria and cells separately, adding bacteria to the cells, and allowing the bacteria and cells to interact;

[0043] In this step, the bacteria are cultured under the combined action of different physical factors and different chemical factors to culture Gram-positive bacteria or Gram-negative bacteria, wherein the physical factor is at least one of bacterial density and fluid shear force, the chemical factor is at least one of reagent type and reagent concentration, the Gram-positive bacteria are at least one of Staphylococcus, Streptococcus and Lactobacillus, and the Gram-negative bacteria are at least one of Escherichia coli, Pseudomonas aeruginosa and Shigella.

[0044] In this step, the cells are cultured under the combined action of different physical factors and different chemical factors, wherein the physical factors are at least one of cell density, extracellular matrix stiffness and geometric constraints of the cells, and the chemical factors are at least one of reagent type and reagent concentration.

[0045] In this step, bacteria are added to the cells to allow the bacteria to interact with the cells, including: culturing the cells in a phenol red-free culture medium containing 10% fetal bovine serum that has been filtered through a 0.22 μm filter membrane under treatment with different chemical factors, wherein the chemical factor is at least one of the type of reagent and the concentration of the reagent, and the filtration is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe; adding bacteria to the cells and allowing the bacteria to interact with the cells at a temperature of 37° C. and a gas environment of 5% CO2 and 95% O2.

[0046] Step S2: Select a fluid force microscope probe and pre-treat the probe;

[0047] In this step, selecting a fluid force microscope probe includes: selecting a fluid force microscope probe with a pinhole diameter of 300nm-1000nm and a spring constant of 0.6-2N / m; wherein the pinhole at the tip of the fluid force microscope probe is smaller than the size of the bacteria and is required to capture a single bacterium to the greatest extent possible, and the spring constant of the fluid force microscope probe is required to be suitable for measuring the adhesion force of cells.

[0048] In this step, the probe is pretreated, including adding a sample solution to the probe sample pool, modifying the probe, and semi-automatically measuring the spring constant k and sensitivity S of the fluid force microscope probe for subsequent adhesion force calculation. The sample solution is a mixture of 10X trypsin and a fluorescent dye that has been centrifuged at 14,800 rpm for 5-20 minutes. The volume ratio of 10X trypsin to fluorescent dye is 3:1, and the centrifugation time can be 10 minutes. High-speed centrifugation of the sample solution prevents impurities in the liquid from clogging the probe pinhole. The trypsin in the sample solution prevents cells from adhering to the probe during measurement, and the fluorescent dye is used to determine whether the probe pinhole is unobstructed. The modification solution is 0.1 mg / ml poly(L-lysine)-grafted-poly(ethylene glycol) that has been centrifuged at 14,800 rpm for 5-20 minutes. The centrifugation time can be 10 minutes, and the immersion time can be greater than 20 minutes. High-speed centrifugation of the modification liquid is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe, and the modification liquid is used to prevent bacteria from irreversibly adhering to the fluid force microscope probe.

[0049] Step S3: searching for a single live bacterium to be captured in the liquid environment where bacteria and cells interact, applying negative pressure using a fluid force microscope pressure controller to cause the pinhole at the probe tip to absorb the bacterium, thereby obtaining a fluid force microscope probe that captures a single bacterium;

[0050] In this step, the negative pressure applied by the pressure controller is -50 to -800 mbar, and the negative pressure enables the pinhole at the tip of the fluid force microscope probe to capture the bacteria.

[0051] Step S4: The fluid force microscope probe that captures a single bacterium is brought close to the cell surface at a preset speed. When a preset force is reached, the probe stays for a preset time and then returns to the probe to obtain adhesion force curve data between the bacteria and the cell.

[0052] In this step, the preset speed is 0.1-5 μm / s, the preset force is 10-100 nN, the preset time is 1-300 s, and the return distance is 20-50 nm.

[0053] Furthermore, after obtaining the adhesion force curve data between bacteria and cells, the method further includes: calculating the adhesion force between bacteria and cells using an adhesion force calculation formula based on the obtained adhesion force curve data between bacteria and cells, wherein the adhesion force calculation formula is:

[0054] F=USk

[0055] Where U(v) is the voltage difference from when the probe is subjected to the adhesion force to when it returns to the initial state, S(m / v) is the probe sensitivity, and k(N / m) is the spring constant.

[0056] Example 1:

[0057] Example 1 provides a method for quantitatively measuring the adhesion force between Staphylococcus aureus transfected with green fluorescent protein and rat small intestinal crypt epithelial cells based on fluid force microscopy, comprising the following steps:

[0058] Step 1: Staphylococcus aureus transfected with green fluorescent protein and rat intestinal crypt epithelial cells (IEC-6 cells) were selected. IEC-6 cells were plated in 12-well plates and cultured in 0.22μm-filtered DMEM medium containing 10% fetal bovine serum (FBS) and lacking phenol red and antibiotics. A small amount of S. aureus was then added to the IEC-6 cells and allowed to interact with the cells at 37°C in an atmosphere of 5% CO₂ and 95% O₂.

[0059] Step 2: Select a fluid force microscope probe and pre-treat the probe.

[0060] The size of the FFM probe's pinhole is selected based on the size of the bacteria. It should be smaller than the bacteria to prevent them from being drawn into the probe and clogging it. Furthermore, the pinhole should not be too small to maximize bacterial capture. Since Staphylococcus aureus has a diameter of approximately 600 nm, a 300 nm diameter was chosen for the FFM probe's pinhole.

[0061] The spring constant of the fluid force microscope probe needs to be suitable for measuring the adhesion force of cells, and the spring constant of the fluid force microscope probe is selected to be 0.6 N / m.

[0062] The pretreatment of the fluid force microscope probe includes: adding sample liquid to the probe sample pool, modifying the probe, and semi-automatically measuring the spring constant and sensitivity of the probe.

[0063] The probe sample pool was filled with 1-1.5 μl of trypsin (10x): fluorescent dye (v / v, 3:1) after high-speed centrifugation (14,800 rpm, 10 minutes). The probe was then mounted in a fluid force microscope and sterilized by immersion in 75% ethanol filtered through a 0.22 μm filter for 1 minute. It was then rinsed in water filtered through a 0.22 μm filter and then immersed in 0.1 mg / ml poly(L-lysine)-graft-poly(ethylene glycol) for 20 minutes after high-speed centrifugation (14,800 rpm, 10 minutes). The spring constant k and sensitivity S of the fluid force microscope probe were semi-automatically measured for subsequent adhesion force calculations.

[0064] Step 3: Allow Staphylococcus aureus to interact with IEC-6 cells in a liquid environment for 15 minutes. The bacteria fall to the surface of the adherent cells under the action of gravity, which facilitates the subsequent capture of the bacteria by the probe. Since Staphylococcus aureus is transfected with green fluorescent protein, the location of Staphylococcus aureus can be clearly observed in the green fluorescence channel of the microscope. Move the fluid force microscope probe to a position approximately 50 μm from the surface of the adherent cells to search for a single Staphylococcus aureus and align the probe pinhole with the Staphylococcus aureus to be captured. Use a pressure controller to apply a negative pressure of -50 mbar to cause the pinhole at the tip of the probe to capture the bacteria, resulting in a fluid force microscope probe that captures a single Staphylococcus aureus.

[0065] Step 4: Schematic diagram of measuring the adhesion force between bacteria and cells based on fluid force microscopy Figure 2 The fluid force microscope probe that captures a single Staphylococcus aureus is moved close to the surface of the adherent cell at a speed of 1 μm / s and stays there for 30 seconds when the applied force reaches 50 nN. The probe then returns to a distance of 20 nm to obtain the adhesion force curve data between the bacteria and the cell. The adhesion force curve data of Staphylococcus aureus and IEC-6 cells are shown in the figure. Figure 3 As shown, the adhesion force between the two is 98.5nN.

[0066] Example 2: This example quantitatively measures the adhesion force between E. coli and IEC-6 cells using a fluid force microscope. The method is basically the same as that of Example 1, except that the bacteria are E. coli transfected with green fluorescent protein. The adhesion force curve data between E. coli and IEC-6 cells are shown in Figure 2. Figure 4 As shown, the adhesion force between the two is 38.3nN.

[0067] Example 3: This example measures the adhesion force between Staphylococcus aureus and IEC-6 cells at different contact times using a fluid force microscope probe. The method is essentially the same as in Example 1, except that the contact times of the fluid force microscope probe are 5s, 10s, 20s, 30s, 40s, 60s, and 120s, respectively. The adhesion force data between Staphylococcus aureus and IEC-6 cells at different contact times using a fluid force microscope probe are shown in Figure 3. Figure 5 shown.

[0068] Example 4: This example measures the adhesion force between Staphylococcus aureus and IEC-6 cells at different moving speeds of the fluid force microscope probe. The method is basically the same as that of Example 1, except that the moving speeds of the fluid force microscope probe are 1 μm / s, 2 μm / s, 3 μm / s, and 4 μm / s, respectively. The adhesion force data between Staphylococcus aureus and IEC-6 cells at different moving speeds of the fluid force microscope probe are shown in Figure 4. Figure 6 shown.

[0069] Example 5: This example uses fluid force microscopy to measure the adhesion force between Staphylococcus aureus and IEC-6 cells under different extracellular matrix stiffnesses. The method is basically the same as that of Example 1, except that IEC-6 cells were cultured on polyacrylamide hydrogels with stiffnesses of 10.14 kPa, 32.29 kPa, and 93.46 kPa, respectively, to allow the cells to be regulated by the extracellular matrix stiffness. The specific steps are as follows:

[0070] Step 1: Activate coverslips (20 mm diameter) to allow adhesion to the polyacrylamide hydrogel. First, wash the coverslips with methanol and dry them at room temperature. Then, activate them with 4% 3-aminopropyltrimethoxysilane, dry them at room temperature, and soak them in distilled water for 30 minutes. Finally, soak the activated coverslips in 0.5% glutaraldehyde for 30 minutes, wash them three times with distilled water, and dry them at room temperature before use.

[0071] Step 2: Pure water, acrylamide, bisacrylamide, ammonium sulfate, and tetramethylethylenediamine were mixed according to the concentrations and proportions shown in Table 1. 25 μl of the mixture was dropped onto the surface of an activated coverslip and covered with a new coverslip (18 mm in diameter). After 20 minutes of polymerization of the mixture at room temperature, the upper clean coverslip was removed to obtain polyacrylamide hydrogels with stiffnesses of 10.14 kPa, 32.29 kPa, and 93.46 kPa, respectively.

[0072]

[0073] Table 1

[0074] Step 3: Incubate type I collagen (0.2 mg / ml) on the surface of the polyacrylamide hydrogel overnight. After removing the collagen, add phosphate buffered saline (PBS) and sterilize under ultraviolet light for 30 minutes. Finally, IEC-6 cells are seeded on the polyacrylamide hydrogels of different stiffness and cultured for 24 hours.

[0075] Step 4: Following the operation of Example 1, the adhesion force between Staphylococcus aureus and cells with different substrate stiffnesses was measured using fluid force microscopy.

[0076] like Figure 7 Figure 2 shows the adhesion force between Staphylococcus aureus and IEC-6 cells measured by fluid force microscopy under different ECM stiffnesses. The adhesion force between bacteria and cells increases with increasing ECM stiffness, indicating that ECM stiffness regulates the interaction between bacteria and cells.

[0077] So far, the present disclosure has been described in detail with reference to the accompanying drawings. Based on the above description, those skilled in the art should have a clear understanding of the present disclosure.

[0078] It should be noted that any implementations not shown or described in the drawings or the main text of the specification are known to those skilled in the art and are not described in detail. Furthermore, the definitions of the components described above are not limited to the specific structures, shapes, or methods described in the embodiments, and can be easily modified or replaced by those skilled in the art.

[0079] Of course, according to actual needs, the present disclosure may also include other parts, which are irrelevant to the innovation of the present disclosure and will not be described here.

[0080] Similarly, it should be understood that in order to streamline the present disclosure and aid in understanding one or more of the various disclosed aspects, in the above description of exemplary embodiments of the present disclosure, various features of the present disclosure are sometimes grouped together into a single embodiment, figure, or description thereof. However, this disclosed approach should not be interpreted as reflecting an intention that the claimed disclosure requires more features than are expressly recited in each claim. Rather, as reflected in the claims below, the disclosed aspects consist of fewer than all the features of the individual embodiments disclosed above. Accordingly, the claims that follow the detailed description are hereby expressly incorporated into the detailed description, with each claim standing on its own as a separate embodiment of the present disclosure.

[0081] In addition, similar or identical parts are numbered the same in the drawings or in the description. The technical features of the various embodiments exemplified in the description can be freely combined to form new solutions, provided there is no conflict. Furthermore, each claim can serve as an embodiment on its own, or the technical features of the various claims can be combined to form new embodiments. Furthermore, while examples of parameters containing specific values ​​may be provided herein, it should be understood that the parameters do not need to be exactly equal to the corresponding values, but can be approximated to the corresponding values ​​within acceptable error tolerances or design constraints.

[0082] Unless there are technical obstacles or contradictions, the above-mentioned various embodiments of the present disclosure can be freely combined to form additional embodiments, and these additional embodiments are all within the protection scope of the present disclosure.

[0083] Although the present disclosure is described in conjunction with the accompanying drawings, the embodiments disclosed in the drawings are intended to illustrate preferred embodiments of the present disclosure and are not to be construed as limiting the present disclosure. The dimensional ratios in the drawings are merely illustrative and are not to be construed as limiting the present disclosure.

[0084] Although some embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined in the claims and their equivalents.

[0085] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above are only specific embodiments of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. A method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy, characterized in that: include: Cultivate bacteria and cells separately, add bacteria to the cells, and allow the bacteria and cells to interact; Select a fluid force microscope probe and pre-treat the probe; Searching for a single live bacterium to be captured in a liquid environment where bacteria and cells interact, using a fluid force microscope pressure controller to apply negative pressure to cause the pinhole at the probe tip to absorb the bacterium, thus obtaining a fluid force microscope probe that captures a single bacterium; as well as The fluid force microscope probe that captures a single bacterium approaches the cell surface at a preset speed. When the preset force is reached, it stays for a preset time and then returns to the probe to obtain the adhesion force curve data between the bacteria and the cell. Among them, in the step of adding bacteria to the cells to allow the bacteria to interact with the cells, the cells are cultured in a phenol red-free culture medium containing 10% fetal bovine serum that has been filtered through a 0.22 μm filter membrane under treatment with different chemical factors, wherein the chemical factor is at least one of the reagent type and the reagent concentration, and the filtration is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe; bacteria are added to the cells and allowed to interact with the cells at a temperature of 37°C and a gas environment of 5% CO2 and 95% O2.

2. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: In the steps of culturing bacteria and cells separately, the culturing of bacteria is culturing Gram-positive bacteria or Gram-negative bacteria under the combined action of different physical factors and different chemical factors, wherein: The physical factor is at least one of bacterial density and fluid shear force; The chemical factor is at least one of the reagent type and the reagent concentration; The Gram-positive bacteria is at least one of Staphylococcus, Streptococcus and Lactobacillus; The Gram-negative bacteria is at least one of Escherichia coli, Pseudomonas aeruginosa and Shigella.

3. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: In the steps of culturing bacteria and cells separately, culturing cells is culturing adherent cells under the combined action of different physical factors and different chemical factors, wherein: The physical factor is at least one of cell density, extracellular matrix stiffness, and geometric constraints of cells; The chemical factor is at least one of the reagent type and the reagent concentration.

4. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: The step of selecting a fluid force microscope probe includes: selecting a fluid force microscope probe with a pinhole diameter of 300nm-1000nm and a spring constant of 0.6-2N / m.

5. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: The step of pre-treating the probe includes: adding sample liquid to the probe sample pool, modifying the probe, and semi-automatically measuring the spring constant k and sensitivity S of the fluid force microscope probe for subsequent calculation of the adhesion force.

6. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 5, characterized in that: The sample solution is a mixed solution of 10X trypsin and a fluorescent dye that has been subjected to high-speed centrifugation at a speed of 14,800 rpm for 5-20 minutes, wherein the volume ratio of 10X trypsin to the fluorescent dye is 3:1; the high-speed centrifugation of the sample solution is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe, the trypsin in the sample solution is used to prevent cells from adhering to the probe during the measurement process, and the fluorescent dye is used to determine whether the probe pinhole is unobstructed; The modification liquid is 0.1 mg / ml poly(L-lysine)-grafted-poly(ethylene glycol) that has been subjected to high-speed centrifugation at a speed of 14,800 rpm for 5-20 minutes, and the immersion time is greater than 20 minutes; high-speed centrifugation of the modification liquid is used to prevent impurities in the liquid from clogging the pinhole of the fluid force microscope probe, and the modification liquid is used to prevent bacteria from irreversibly adhering to the fluid force microscope probe.

7. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: In the step of using a fluid force microscope pressure controller to apply negative pressure to cause the pinhole at the probe tip to absorb bacteria, the negative pressure applied by the pressure controller is -50 to -800 mbar, and the negative pressure enables the pinhole at the probe tip of the fluid force microscope to capture bacteria.

8. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: In the step of bringing the fluid force microscope probe for capturing a single bacterium close to the cell surface at a preset speed, staying for a preset time when reaching a preset force, and then returning to the probe to obtain adhesion force curve data between the bacteria and the cell, the preset speed is 0.1-5 μm / s, the preset force is 10-100 nN, the preset time is 1-300 s, and the return distance is 20-50 nm.

9. The method for quantitatively measuring the adhesion force between bacteria and cells based on fluid force microscopy according to claim 1, characterized in that: After obtaining the adhesion force curve data between bacteria and cells, the method further includes: Based on the obtained adhesion force curve data between bacteria and cells, the adhesion force between bacteria and cells was calculated using the adhesion force calculation formula, which is: Where U (v) is the voltage difference from when the probe is subjected to adhesion force to when it returns to its initial state, S (m / v) is the probe sensitivity, and k (N / m) is the spring constant.

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