Bacteria probe as well as preparation method and application thereof
The two-step method for preparing bacterial probes solves the problems of uncontrollable probe position, short survival time and complicated operation in existing technologies, and realizes bacterial probes with high biological activity and mechanical stability, which are suitable for measuring the interaction force between bacteria and samples.
Patent Information
- Application Number
- CN202511291406.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-10
- Publication Date
- 2026-01-27
AI Technical Summary
Existing AFM bacterial probe preparation methods suffer from problems such as uncontrollable bacterial growth location, short bacterial survival time, high operational difficulty, and cantilever contamination.
A two-step method was used to prepare bacterial probes, which involved modifying microspheres in a bacterial adhesion-enhancing modifier and culturing bacteria on the surface of the microspheres. The bacteria were then brought into contact with an atomic force microscope probe cantilever beam, resulting in bacterial probes with good uniformity and high reproducibility.
It achieves high bioactivity and mechanical stability of bacterial probes, extends single-probe testing time, improves reusability, and provides a precise tool for measuring adhesion force, suitable for research on adhesion mechanisms in multiple scenarios.
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Figure CN121406477A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microbiology, and particularly relates to a bacterial probe and a preparation method and application thereof. BACKGROUND
[0002] The formation of multicellular microbial communities (biofilms) begins with the adhesion of planktonic bacteria to surfaces, which is synergistically regulated by environmental factors (such as nutrition, pH), surface properties (such as roughness, chemical composition), and bacterial characteristics (such as pili, extracellular polysaccharides). The transition from reversible adhesion to irreversible colonization is a key step in the development of biofilms, involving physical and chemical processes such as van der Waals forces, electrostatic interactions, and specific molecular recognition. Due to the significant impact of biofilms in medical infections (such as contamination of implanted devices), industrial equipment fouling, and environmental remediation, understanding the microscopic mechanical mechanisms of bacterial adhesion has significant scientific and application value.
[0003] To study the adhesion mechanism of bacteria, traditional methods such as plate and washing method, rotating disc method, and centrifugation method can only achieve qualitative or semi-quantitative analysis, and cannot accurately quantify the adhesion force at the single cell level. Optical tweezers and nanoindentation technology can measure force, but are limited by the complexity of operation and resolution. Atomic force microscopy (AFM) has become the main technology for directly measuring the adhesion force between bacteria-surface, bacteria-bacteria, and bacteria-host cells under physiological conditions, due to its piconewton force resolution and nanometer spatial accuracy. AFM force curve testing can cover a wide range of 10 pN to 1 μN and 1 nN to 10 μN, suitable for analyzing various adhesion interactions from weak van der Waals forces to strong chemical bonding.
[0004] However, the preparation of AFM bacterial probes is the key to achieving accurate adhesion force measurement. Current mainstream methods include: 1) direct glue fixation method: bacteria are adhered to the end of the cantilever by epoxy resin or biological glue. This method is simple to operate, but chemical cross-linking can cause bacterial death, making it impossible to study the dynamic adhesion behavior of living cells; 2) cantilever adsorption method: the cantilever is immersed in the bacterial solution, relying on physical adsorption or specific ligand-receptor interaction to capture bacteria. This method is prone to contamination of the cantilever, and bacteria are randomly distributed on the cantilever surface, making it difficult to locate target cells; 3) bacterial cluster growth method: bacterial clusters are cultured on the cantilever surface. This method has the problems of uncontrollable bacterial growth location, high heterogeneity of cells within the cluster, and uneven mechanical properties, resulting in poor repeatability.
[0005] Therefore, the existing preparation methods of AFM bacterial probes have the problems of uncontrollable bacterial growth location, short bacterial survival time, high operation difficulty, and cantilever contamination. SUMMARY
[0006] The purpose of this application is to provide a method for preparing bacterial probes, which aims to solve the problems of uncontrollable bacterial growth location, short bacterial survival time, high operational difficulty, and cantilever contamination in existing AFM bacterial probe preparation methods.
[0007] The embodiments of this application are implemented as follows: a method for preparing a bacterial probe includes:
[0008] The microspheres were placed in a bacterial adhesion-enhancing modifier for surface modification for no less than 4 hours;
[0009] Bacterial suspension was added to surface-modified microspheres for cultivation and growth treatment to ensure that bacteria grow uniformly on the surface of the microspheres. After centrifugation and washing, bacterial microspheres were obtained.
[0010] The bacterial microspheres are dispersed by shaking and then brought into contact with an atomic force microscope probe cantilever beam coated with glue, so that they adhere to the atomic force microscope probe cantilever beam to obtain a bacterial probe.
[0011] Another objective of this application is to provide a bacterial probe prepared by the above-described method for preparing bacterial probes.
[0012] Another objective of this application is the application of the above-described bacterial probe in measuring the interaction force between bacteria and a sample.
[0013] This application presents an innovative two-step method for preparing uniform, reproducible, and highly viable bacterial probes, successfully solving the problems of low survival rate, uncontrollable positioning, and complex operation in existing AFM bacterial probe preparation methods. This method achieves precise control of bacterial colonization location and uniform bacterial probe preparation through a microsphere modification-bacterial growth-probe preparation approach, avoiding contamination of the cantilever and laser signal interference from traditional underwater operations. The bacterial probes prepared in this application exhibit excellent encapsulation properties, combining high biological activity (survival in air >20 hours) with mechanical stability, significantly extending single-probe testing time and reusability. Furthermore, controllable insertion depth enables targeted contact between bacteria and the substrate, providing a reliable tool for obtaining real adhesion force data and bacterial mechanical properties. This technology offers batch preparation capabilities and ease of operation, applicable to research on bacterial-surface and bacterial-cell adhesion mechanisms in various scenarios, providing an efficient biomechanical analysis method for biofilm formation, infection control, and other fields. Attached Figure Description
[0014] Figure 1 This is a flowchart illustrating the preparation process of bacterial microspheres provided in the embodiments of this application;
[0015] Figure 2 A schematic diagram illustrating the preparation of bacterial probes is provided for embodiments of this application;
[0016] Figure 3 SEM images of bacterial microspheres obtained under different bacterial adhesion-enhancing modifiers, different incubation times, and different washing cycles, as provided in the embodiments of this application;
[0017] Figure 4 Fluorescence spectra of bacterial microspheres obtained by modification with different bacterial adhesion-enhancing modifiers, as provided in the embodiments of this application;
[0018] Figure 5 Fluorescence spectrum of bacteria on bacterial microspheres provided in this application embodiment after being placed in air for 15 hours;
[0019] Figure 6 The fluorescence spectrum of bacteria on bacterial microspheres after bacterial death is provided in the embodiments of this application;
[0020] Figure 7 SEM images of bacterial microspheres and bacterial probe force curves are provided for embodiments of this application.
[0021] Figure 8 The bacterial probe mechanical testing mapping diagram and adhesion force distribution diagram provided in the embodiments of this application;
[0022] Figure 9 The following are examples of measurements of Young's modulus of Escherichia coli using different probes, along with a model and a comparison chart of Young's modulus, provided for embodiments of this application.
[0023] Figure 10 Fluorescence spectra of bacterial microspheres obtained at different incubation times, as provided in the embodiments of this application;
[0024] Figure 11 SEM images of bacterial microspheres obtained at different centrifugation speeds and different dispersion methods, provided for the embodiments and comparative examples of this application. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.
[0026] To address the challenges of low survival rate, uncontrollable placement, and complex operation in existing AFM bacterial probe preparation methods, this application proposes a two-step method for preparing uniform, reproducible, and highly viable bacterial probes. The method involves microsphere modification, bacterial growth, and probe preparation, attaching bacteria to PS microspheres to prepare probes for atomic force microscopy (AFM) applications. The bacterial probes prepared using this method exhibit excellent encapsulation, effectively extending bacterial survival time and thus testing time. Furthermore, the contact position between bacteria and the sample can be accurately controlled, improving experimental reproducibility. These bacterial probes can be used for measuring bacterial adhesion forces and studying bacterial adhesion mechanisms. The preparation of these bacterial probes is highly operable, controllable, and does not require submersible operation, making them a valuable tool for biomechanical testing that warrants widespread application.
[0027] Specifically, embodiments of this application provide a method for preparing a bacterial probe, comprising:
[0028] The microspheres were placed in a bacterial adhesion-enhancing modifier for surface modification for no less than 4 hours;
[0029] Bacterial suspension was added to surface-modified microspheres for cultivation and growth treatment to ensure that bacteria grow uniformly on the surface of the microspheres. After centrifugation and washing, bacterial microspheres were obtained.
[0030] The bacterial microspheres are dispersed by shaking and then brought into contact with an atomic force microscope probe cantilever beam coated with glue, so that they adhere to the atomic force microscope probe cantilever beam to obtain a bacterial probe.
[0031] In the embodiments of this application, the material of the microspheres can be selected from polystyrene (PS) (Jiangsu Zhichuan Technology), silicon dioxide (SiO2) (Jiangsu Zhichuan Technology), silicon nitride (Si3N4) (Shanghai Bruker Technology Co., Ltd.), or glass. The diameter of the microspheres is between 2-20 μm.
[0032] In the embodiments of this application, the bacteria used for testing can be Gram-positive bacteria (such as Staphylococcus aureus) or Gram-negative bacteria (such as Escherichia coli), and are derived from the Guangdong Microbial Culture Collection Center. The number of passages of the bacteria used is within 2 generations.
[0033] In this embodiment, the bacterial suspension can be obtained by the following conventional method: 100 μL of bacterial suspension in the exponential growth phase is inoculated into a centrifuge tube containing 10 mL of M9 solution, and cultured at 25°C for 24 h with a shaker speed of 150 rpm until the concentration reaches OD200. 600nm≈0.6-0.8. Then, centrifuge at 5000 r / min for 5-10 min, remove the supernatant, and then add 10 mL of PBS buffer to the centrifuge tube to resuspend the bacteria. Centrifuge at 5000 r / min for 5-10 min. Repeat this step three times to obtain a bacterial suspension in PBS.
[0034] In the embodiments of this application, the bacterial adhesion-enhancing modifier may be polydopamine (PD) (Aladdin reagent), polylysine (PLL) (Aladdin reagent), polyornithine (PO), or cell adhesion agent (Cell-Tak). TM One or more of the following, more preferably polydopamine. A layer of functional groups conducive to bacterial growth is attached to the surface of the microspheres, and the residue is then washed away to obtain a microsphere substrate suitable for bacterial growth.
[0035] Optionally, the bacterial adhesion-enhancing modifier has no inactivating effect on bacterial growth. Taking polydopamine as an example, excessive polydopamine concentration can lead to bacterial death, while insufficient concentration cannot guarantee strong bacterial adhesion to the microspheres. Therefore, it is necessary to control its concentration (2-15 mg / mL) and perform washing after modification to remove surface residues. Dopamine hydrochloride is used as a precursor at a concentration of 2-15 mg / mL, and 2-10 mmol / L of tris(hydroxymethyl)aminomethane is added as a buffer. Sodium hydroxide is added as a regulator to adjust the pH to 6.5-8.5. The microspheres are modified in the dark for 4-10 hours. Furthermore, modification time less than 4 hours will weaken the viscosity effect of bacteria on the PS microspheres, making it impossible to obtain completely encapsulated bacterial microspheres, and bacteria may detach during testing.
[0036] In this embodiment of the application, the step of placing the microspheres in the bacterial adhesion enhancement modifier for surface modification for no less than 4 hours can be as follows: take 20-100 μL of polystyrene microsphere dispersion with a solid content of 5% and add it to a centrifuge tube, prepare a bacterial adhesion enhancement modifier with a concentration of 2-15 mg / mL, take 1 mL of the bacterial adhesion enhancement modifier and add it to the centrifuge tube, place it in the dark for modification for 4-10 hours, and then wash it with deionized water at 5000 r / min to remove the residual modifier on the surface, and obtain the surface-modified microspheres.
[0037] In this embodiment, the step of adding the bacterial suspension to the surface-modified microspheres for cultivation and growth treatment to ensure uniform bacterial growth on the microsphere surface, followed by centrifugation and washing to obtain bacterial microspheres, can be as follows: Add the bacterial suspension to the surface-modified microspheres for cultivation and growth treatment to ensure uniform bacterial growth on the microsphere surface, and centrifuge and wash 1-2 times at 3000-4500 r / min to obtain bacterial microspheres. Specifically, this can be done by: adding 100-500 μL of bacterial suspension to the surface-modified microspheres, then adding 1 mL of broth, and cultivating at room temperature for 10-14 days to ensure uniform and firm bacterial growth on the microsphere surface. Then, centrifuge at 6000 r / min for 5 minutes to remove the broth solution, and then centrifuge and wash once with PBS buffer at 4000 r / min to remove residual broth, non-adherent bacteria, unattached bacterial microspheres, and incompletely coated bacterial microspheres, resulting in well-coated bacterial microspheres.
[0038] In this embodiment, the step of dispersing the bacterial microspheres by shaking and then contacting them with an atomic force microscope (AFM) probe cantilever beam coated with adhesive to obtain the bacterial probe cantilever beam can be performed as follows: Add PBS buffer to the bacterial microspheres and disperse by shaking (e.g., add 1 mL of PBS buffer and shake for 1 min) to obtain a bacterial microsphere dispersion; control the AFM probe cantilever beam to lightly touch the adhesive layer so that the tail end of the AFM probe cantilever beam is coated with adhesive; drop 20-80 μL of the bacterial microsphere dispersion onto the surface of a mica sheet; control the AFM probe cantilever beam coated with adhesive to focus directly above the bacterial microspheres and lower the probe so that the adhesive layer at the tail end of the AFM probe cantilever beam contacts the bacterial microspheres; maintain this position for 30-150 s, then lift the AFM probe cantilever beam and leave it in the air for 25-60 min to obtain the bacterial probe.
[0039] Optionally, to investigate the effect of preparation process parameters on the obtained bacterial microspheres, this application adopts the following methods, and the preparation process can be referred to... Figure 1 As shown, the microspheres were modified in dopamine hydrochloride solution for 1-10 hours, washed 1-3 times by centrifugation with deionized water at 5000-8000 r / min, and 2 mL of a mixture of broth and bacterial suspension was added (the volume ratio of bacterial suspension to broth was 1:3). The mixture was incubated at 15-30℃ for several days, and then washed by centrifugation with PBS buffer at speeds of 6000 r / min, 5000 r / min, 4000 r / min, and 3000 r / min to obtain the bacterial probe. The research results are shown in the following specific examples.
[0040] In this embodiment, the spring constant K of the atomic force microscope probe cantilever beam is 16-40 N / m, such as 16 N / m, 25 N / m, 40 N / m, etc. A K value that is too small (K < 16) will cause the measurement result to exceed the range, while a K value that is too large (K > 40) will lead to a decrease in measurement accuracy.
[0041] This application also provides a bacterial probe, which is prepared by the above-described method for preparing bacterial probes.
[0042] This application also provides an application of the above-mentioned bacterial probe in measuring the interaction force between bacteria and a sample. The application method includes: adjusting the laser feedback signal SUM value on the bacterial probe to its maximum to ensure maximum force transmission sensitivity; calibrating the probe cantilever laser sensitivity (Deflection Sensitivity, nm / V) on a hard standard sample; and calibrating the probe's spring constant K (N / m) using the Sadar method. Then, the sample to be tested is placed on the sample stage, and the stage is moved to position the sample directly above the bacterial probe microspheres for insertion. The indentation voltage is set to 2V, the ramp rate to 1μm / s, and 100 force curves are obtained by sequentially testing in 10 regions.
[0043] The preparation method of bacterial probes is described in detail below with specific embodiments, as shown below. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0044] Example 1: Preparation of bacterial probes
[0045] 100 μL of E. coli suspension in the exponential growth phase was inoculated into a centrifuge tube containing 10 mL of M9 solution and cultured at 25 °C for 24 h at a shaker speed of 150 rpm until the concentration reached OD100. 600nm ≈0.7. Then, centrifuge at 5000 r / min for 5 min, remove the supernatant, and then add 10 mL of PBS buffer to the centrifuge tube to resuspend the bacteria. Centrifuge at 5000 r / min for 5 min. Repeat this step three times to obtain an E. coli suspension.
[0046] Prepare a 4 mg / mL dopamine hydrochloride solution, add 10 mmol / L tris(hydroxymethyl)aminomethane, adjust the pH to 8.5, use a pipette to measure 100 μL of 2.5% solids PS microspheres (10 μm in diameter) dispersion and add it to the above solution. Modify in the dark for 4 h, centrifuge at 6000 r / min and wash three times with deionized water.
[0047] Add 200 μL of E. coli suspension and 1 mL of broth nutrient solution to the cleaned PS microspheres, and incubate at 25°C and 55% RH for 14 days to allow the bacteria to grow uniformly and firmly on the microsphere surface. Take 1 mL of the incubated microsphere bacterial suspension and place it in a 2 mL centrifuge tube. Centrifuge at 6000 rpm for 5 min to remove the broth solution, then centrifuge once with PBS buffer at 4000 rpm to obtain E. coli microspheres. Add 1 mL of PBS buffer to the E. coli microspheres to disperse the precipitate, and shake for 1 min to obtain an E. coli microsphere dispersion.
[0048] like Figure 2 As shown, UHU adhesive (Dongguan Yihui Adhesive Co., Ltd.) from Germany was mixed with adhesive A and adhesive B in a 1:1 mass ratio and a thin layer of adhesive was evenly applied to a glass slide. The TL-NCH cantilever beam (Hechuang Science & Technology Co., Ltd.) was assembled in the probe holder and installed in the AFM scanning tube. The laser feedback signal SUM value was adjusted, and the beam was positioned at the end of the cantilever beam where the bacterial microspheres were to be bonded. The cantilever beam was controlled to focus on the edge of the adhesive layer. The probe was then inserted at the edge of the adhesive layer, lightly touching it to adhere a thin layer of adhesive to the end of the cantilever beam. The cantilever beam was then inserted into a blank area of the slide, and the Ramp function was used to gently press the slide surface with a 1V voltage to remove excess adhesive until a small amount remained sufficient to support the adhesion of the bacterial microspheres. The probe was then lifted. Take 60 μL of E. coli microsphere dispersion and drop it onto a clean mica sheet. Control the probe cantilever to focus directly above the E. coli microspheres and insert the probe. Use the Ramp function to make the glue layer at the end of the cantilever contact the bacterial microspheres. Hold for 30 seconds and then lift the probe. Leave it in the air for 30 minutes to allow the glue to solidify, and you will get the bacterial probe. Store the bacterial probe in a box containing PBS solution.
[0049] Furthermore, 100 μL of the *E. coli* microsphere dispersion prepared in Example 1 was subjected to SEM characterization. The bacteria were fixed with 2.5% glutaraldehyde solution at 4°C for 4 h, and then successively replaced with 10%, 30%, 50%, 70%, 90%, and 95% ethanol solutions for 15 min each time, followed by centrifugation. Then, anhydrous ethanol was used for replacement three times, 15 min each time. The SEM results are as follows. Figure 3 As shown in (a), it can be seen that Escherichia coli is uniformly and completely coated on the surface of PS microspheres.
[0050] Furthermore, 100 μL of the *E. coli* microsphere dispersion prepared in Example 1 was diluted to 1 mL with PBS, stained with STYO9-PI live / dead bacteria staining agent, and observed under a laser confocal fluorescence microscope to verify bacterial viability. The results are as follows: Figure 4 As shown in (a). The survival of bacteria was observed by placing the bacteria in the air for a certain period of time, and the results are as follows.Figures 5-6 As shown, Figure 5 This demonstrates that after being exposed to air for 15 hours, the bacterial survival rate remained above 80%. Figure 6 This proves that the bacteria remained attached to the PS microspheres even after they died.
[0051] Furthermore, a bacterial probe was installed in the AFM, the laser position was adjusted to strengthen the feedback signal, and a force curve was obtained by pressing it into a flat location on the mica sheet. The laser signal sensitivity Z-sensitivity was calculated, and the cantilever spring constant K was calculated using the Sader method. Specifically, Z-sensitivity = 64.5 nm / V, and K = 23.8 N / m. Figure 7 (ad) shows a SEM image of bacterial microspheres prepared from PS microspheres, and then bacterial probes obtained by adhering the bacteria to an empty suspension wall. Figure 7 (e) shows that the bacterial probe force curves maintained high overlap after 24 hours, supporting long-term testing. Meanwhile... Figure 8 The mapping experiment (af) showed good adhesion overlap after testing 256 force curves consecutively, proving that the bacterial probe did not change during the testing process. Figure 8 (g) shows the distribution of adhesion force in the Mapping test.
[0052] Place the mica sheet on the sample stage and move the stage so that the mica sheet is directly above the bacterial probe for insertion. Set the indentation voltage to 2V and the ramp rate to 1μm / s. Select 10 points on the mica sheet sequentially, and measure 10 force curves at each point, obtaining 100 force curves. Repeat the steps with different probes (PS colloidal microsphere probe, cone probe) to obtain 100 force curves on each material. The results of measuring Young's modulus of E. coli with different probes and comparing it with the model and Young's modulus are as follows. Figure 9 As shown, the bacterial probe provided in this application has a smaller error in measuring the Young's modulus of bacteria compared to other methods or probes.
[0053] Example 2: Preparation of bacterial microspheres using different bacterial adhesion-enhancing modifiers
[0054] Based on Example 1 above, the bacterial adhesion-enhancing modifier was replaced with polylysine (PLL), and the resulting E. coli microsphere dispersion was characterized by SEM and fluorescence. The test results are as follows: Figure 3 (b) and Figure 4 As shown in (b). Wherein, from Figure 3 As can be seen from the SEM image of (ac), after 14 days of PD-modified culture and one wash, the bacteria completely adhered to and encapsulated the microspheres. Figure 3 (a)】After culturing with PLL-modified microspheres for 14 days and washing once, the bacteria partially adhered to the microspheres. Figure 3(b)】, and unmodified bacteria cannot adhere to and encapsulate microspheres
Figure 3 (c)
Figure 4 (c)
[0055] Example 3: Preparation of bacterial microspheres under different incubation times
[0056] Based on Example 1 above, the incubation time in the step "adding 200 μL of E. coli suspension and 1 mL of broth nutrient solution to the cleaned PS microspheres, and incubating at 25°C and 55% RH for 14 days to allow the bacteria to grow uniformly and firmly on the surface of the microspheres" was adjusted to 7 days and 20 days, respectively. The obtained E. coli microsphere dispersions were characterized by SEM and fluorescence. The test results are as follows: Figure 3 (d, f) and Figure 10 As shown in (a) and (c). From Figure 3 As can be seen from the SEM image (df), the bacterial microspheres obtained by using PD as a modifier and culturing for 7 days after one wash exhibit partial adhesion. Figure 3 (d)】, Bacterial microspheres obtained after 14 days were completely adhered to [ Figure 3 (e)】The bacterial microspheres obtained after 20 days were completely adhered to [ Figure 3 (f)】。From Figure 10 As can be seen from the fluorescence image (ac), after 7 days of culture using PD as a modifier, partial adhesion and complete bacterial survival were observed. Figure 10 (a)】, 14 days later, complete adhesion and partial bacterial death were observed. Figure 10 (b) 20 days later, most bacteria died. Figure 10 (c)】
[0057] Example 4: Preparation of bacterial microspheres under different washing cycles
[0058] Based on Example 1 above, the number of washes in the step "washing once with PBS buffer at a speed of 4000 r / min to obtain E. coli microspheres" was adjusted to 0 and 3 times respectively, and the obtained E. coli microsphere dispersion was characterized by SEM. The test results are as follows: Figure 3 As shown in (g, i). From Figure 3 As can be seen from (gi), there is broth residue in the unwashed 14-day-old culture. Figure 3 (g)】, no residue after one wash
Figure 3(h)
Figure 3 (i)
[0059] Example 5: Preparation of bacterial microspheres at different centrifugation speeds
[0060] Based on Example 1 above, the centrifugation speed in the step "washing once with PBS buffer at 4000 r / min to obtain E. coli microspheres" was adjusted to 6000 r / min, 5000 r / min, 4000 r / min, 3000 r / min, 2500 r / min, and 2000 r / min, respectively. The obtained E. coli microsphere dispersion was characterized by SEM, and the test results are as follows: Figure 11 As shown in (af), centrifugation speeds of 6000 r / min and 5000 r / min resulted in bacterial microspheres sticking together, exhibiting poor dispersion and the presence of unattached bacteria; centrifugation speeds below 3000 r / min resulted in some microspheres that were not completely coated with and adhered to bacteria.
[0061] Comparative Example 1:
[0062] Based on Example 1 above, the step "add 1 mL of PBS buffer to E. coli microspheres to disperse the precipitate, and shake with a shaker for 1 min to obtain E. coli microsphere dispersion" was adjusted from shaking dispersion to ultrasonic dispersion, and the obtained E. coli microsphere dispersion was characterized by SEM. The test results are as follows: Figure 11 As shown in (gi), it can be seen that the undispersed bacterial microspheres are completely adhered together. Figure 11 (g)】, shaken to disperse to obtain individual bacterial microspheres
Figure 11 (h)
[0063] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
[0064] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for preparing a bacterial probe, characterized in that, include: The microspheres were placed in a bacterial adhesion-enhancing modifier for surface modification for no less than 4 hours; Bacterial suspension was added to surface-modified microspheres for cultivation and growth treatment to ensure that bacteria grow uniformly on the surface of the microspheres. After centrifugation and washing, bacterial microspheres were obtained. The bacterial microspheres are dispersed by shaking and then brought into contact with an atomic force microscope probe cantilever beam coated with glue, so that they adhere to the atomic force microscope probe cantilever beam to obtain a bacterial probe.
2. The method for preparing a bacterial probe according to claim 1, characterized in that, The bacterial adhesion-enhancing modifiers are polydopamine, polylysine, polyornithine, and Cell-Tak. TM One or more of 3-aminopropyltriethoxysilane.
3. The method for preparing a bacterial probe according to claim 2, characterized in that, The bacterial adhesion-enhancing modifier is polydopamine.
4. The method for preparing a bacterial probe according to claim 1, characterized in that, The step of placing the microspheres in a bacterial adhesion-enhancing modifier for surface modification for no less than 4 hours includes: Add 20-100 μL of polystyrene microsphere dispersion with a solid content of 5% to a centrifuge tube, prepare a bacterial adhesion enhancement modifier with a concentration of 2-15 mg / mL, add 1 mL of bacterial adhesion enhancement modifier to the centrifuge tube, and place it in the dark for 4-10 hours for modification. Then wash with deionized water at 5000 r / min to remove residual modifier on the surface, and obtain surface-modified microspheres.
5. The method for preparing a bacterial probe according to claim 1, characterized in that, The step of adding the bacterial suspension to surface-modified microspheres for cultivation and growth treatment to ensure uniform bacterial growth on the surface of the microspheres, followed by centrifugation and washing to obtain bacterial microspheres, includes: The bacterial suspension was added to the surface-modified microspheres for cultivation and growth treatment to ensure that the bacteria grew uniformly on the surface of the microspheres. The microspheres were then centrifuged and washed 1-2 times at a speed of 3000-4500 r / min to obtain bacterial microspheres.
6. The method for preparing a bacterial probe according to claim 5, characterized in that, The step of adding the bacterial suspension to surface-modified microspheres for cultivation and growth treatment to ensure uniform bacterial growth on the microsphere surface, and then centrifuging and washing 1-2 times at a speed of 3000-4500 r / min to obtain bacterial microspheres includes: Add 100-500 μL of bacterial suspension to the surface-modified microspheres, then add 1 mL of broth and incubate at room temperature for 10-14 days to allow the bacteria to grow evenly on the surface of the microspheres. Then centrifuge at 6000 r / min for 5 minutes to remove the broth solution, and then centrifuge once with PBS buffer at 4000 r / min to obtain bacterial microspheres.
7. The method for preparing a bacterial probe according to claim 1, characterized in that, The step of dispersing the bacterial microspheres by shaking and then contacting them with an atomic force microscope probe cantilever beam coated with adhesive to adhere them to the cantilever beam, thereby obtaining a bacterial probe, includes: PBS buffer was added to the bacterial microspheres and the mixture was shaken to disperse them, resulting in a monodisperse bacterial microsphere dispersion. Control the atomic force microscope probe cantilever beam to lightly touch the glue layer so that glue adheres to the tail end of the atomic force microscope probe cantilever beam; Take 20-80 μL of monodisperse bacterial microsphere dispersion and drop it onto the surface of a mica sheet. Control the atomic force microscope probe cantilever beam, which is coated with adhesive, to focus directly above the bacterial microspheres and insert the probe so that the adhesive layer at the end of the atomic force microscope probe cantilever beam contacts the bacterial microspheres. After holding this position for 30-150 seconds, lift the atomic force microscope probe cantilever beam and leave it in the air for 25-60 minutes to obtain the bacterial probe.
8. The method for preparing a bacterial probe according to claim 1, characterized in that, The spring constant K of the atomic force microscope probe cantilever beam is 16-40 N / m.
9. A bacterial probe, characterized in that, The bacterial probe is prepared by the method described in any one of claims 1-8.
10. The use of the bacterial probe of claim 9 in measuring the interaction force between bacteria and a sample.