CN113267545B

CN113267545B - In-situ test system and test method for quantitatively detecting adhesion behavior of single living bacteria

Info

Publication number: CN113267545B
Application number: CN202110564386.9A
Authority: CN
Inventors: 和庆钢; 张硕猛; 王磊; 李中坚; 邵文怡
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2023-05-02
Anticipated expiration: 2041-05-24
Also published as: CN113267545A

Abstract

The invention discloses an in-situ test system and a test method for quantitatively detecting the adhesion behavior of single living bacteria, wherein the test system comprises an electromagnetic shielding box, an electrochemical workstation, a computer, a damping table arranged in the electromagnetic shielding box, and an atomic force microscope and an optical inversion microscope which are arranged on the damping table; the atomic force microscope comprises an AFM base plate and an AFM scanner. The system and the method can quantitatively detect the adhesion behavior (including the adhesion between bacteria and the material surface, the length of a biological macromolecular chain of an extramembranous protein, the average number of unfolding events and the average unfolding force) of single living bacteria on the material surface in the real physiological metabolism process of the bacteria, and simultaneously obtain an electronic transfer signal between the single living bacteria and the material.

Description

In-situ test system and test method for quantitatively detecting adhesion behavior of single living bacteria

Technical Field

The invention belongs to the field of microorganisms, and relates to an in-situ test system and a corresponding in-situ test technical method thereof, which can be used for quantitatively detecting the real adhesion behavior of single living bacteria on the surface of a material under the normal metabolism condition of the bacteria.

Background

Bacteria are one of the main groups of organisms on earth, and are the most numerous of all organisms. It is widely distributed in nature or symbiotic with other organisms. The human body also has a considerable amount of bacteria. It is estimated that the total number of bacterial cells in and on the human body is about ten times the total number of cells in the human body. In addition, some species are also distributed in extreme environments, such as spas, and even radioactive waste. Bacteria irreversibly attach to the surface of living or inanimate entities, multiply, differentiate, and secrete some polysaccharide matrix, encapsulating the bacterial population therein to form a film of bacterial aggregates, known as a biofilm. Biofilms are widely present in nature and in living beings, and the formation of biofilms brings significant economic losses for human social production and seriously threatens human health and even life safety. Instrument implantation is one of the most widely used treatments in modern clinical medicine, and is aimed at providing internal support for biological tissues to achieve corresponding therapeutic effects, and is widely used in orthopaedics, cardiovascular and dental surgery, for example: implanted steel plates, bone nails, catheters, cardiac pacemakers, dental braces and the like. However, bacteria are very prone to adhere to the surface of these indwelling implants and form biofilms, causing infections and corresponding complications. And the biofilm is difficult to remove once formed, because bacteria in the biofilm secrete a large amount of extracellular polymers such as proteins, polysaccharides, peptidoglycans, lipids, phospholipids and the like, so that a protective layer is provided for the bacteria, the bacteria are not easy to cure even if large-dose antibiotics are used, the bacteria are extremely easy to avoid the killing of host immune mechanisms, the bacteria possibly develop into refractory infections and even form sepsis, and the life health of a human body is seriously threatened. The infected implantation instrument often needs to be removed again for thorough cleaning by surgery or needs to be replaced with a new implant, which brings great pain to the patient and also brings great economic burden. In addition, steels play a vital role in the development of the modern industry, including: ships, bridges, pipes, rails, etc., and are thus referred to as "industrial bones". However, steel corrosion brings huge economic losses to the country every year. Wherein microbial corrosion (MIC) is one of the major forms of corrosion of steel. Microbial corrosion can directly or indirectly affect the corrosion process of metallic materials by relying on the biological activity of the microorganisms themselves and their metabolic corrosive effects. Bacteria can take electrons directly from the metal surface to accelerate the dissolution of the metal, with the aim of obtaining energy. The bacteria can directly accelerate the corrosion of the metal matrix through enrichment and growth under the biological film, and the corrosion caused by the bacteria accounts for more than 20 percent of the total corrosion, so that the economic loss is huge.

The biomembrane formed by bacteria can be controllably utilized by people while bringing economic loss and life health threat, and brings corresponding economic benefit and technical result for human beings. Environmental pollution is another troublesome problem that needs to be faced in the current world, and the problem of water environment pollution is particularly serious. In the bead triangle and long triangle areas with developed economy, the problem of industrial wastewater discharge is serious due to a large number of enterprises such as pharmacy, printing and dyeing, papermaking and the like. However, conventional sewage treatment systems require the consumption of large amounts of fossil fuels, which in turn exacerbates the energy pressures. From another point of view, industrial wastewater contains a large amount of organic matters, and belongs to renewable and low-pollution biomass energy. This has important implications for both energy shortage and environmental pollution if it is possible to recover this energy from the wastewater and convert it into usable energy. The microbial electrochemical system (BES) is a research hotspot in the fields of energy and environment, combines microbiological, electrochemical, environmental engineering, materialy and other subjects, and cross-merges a green resource recycling technology. BES uses bacteria as a catalyst to form a biofilm on the electrode surface, which breaks down degradable organics in wastewater through intrinsic respiratory chains and converts the energy obtained into electrical or other chemical energy. The technology provides a new idea for changing the energy structure and promoting the environmental treatment. In addition, technical efforts such as microbial sensors and microbial synthesis have been developed by using biological membranes.

Therefore, the biological film is a double-edged sword, and the formation of harmful biological film needs to be inhibited, so that the development of beneficial biological film is promoted. At present, a great deal of research is being conducted to inhibit the formation of biofilms, including: developing a material surface antibacterial coating, developing a powerful bactericide, designing a novel antibacterial material, and the like. In order to promote the formation of a biofilm, attempts have been made to develop novel biocompatible materials, design material surface morphology structures, and the like. However, biofilm formation is still not effectively controlled. This is due to the lack of adequate knowledge of the mechanism of biofilm formation, which has led to a material development and structural design that remains empirical. By conducting extensive research on the special phenomenon that bacteria play a dominant role in solid surface colonization, it is increasingly recognized that the formation of these biofilms involves complex physicochemical processes and interactions of biological communities, which present a great challenge for biofilm research. The formation of a biofilm on the surface of a material is a complex, dynamic process, mainly comprising 5 steps: bacterial adhesion, growth, maturation, dispersion and remodeling. Among these, adhesion of bacteria to the surface of the material is the first step in biofilm formation and is also the most important step. Thus, the study of the adhesion behavior of bacteria on the surface of materials will help to deepen our understanding and understanding of the mechanism of biofilm formation. In traditional researches, researchers adopt plate-and-wash experiments, bacterial force experiments, centrifugal separation experiments, flow chamber experiments and well plate experiments to mainly account for the adhesion performance of bacteria on the surface of a material by counting the number or proportion of bacteria on the surface of the material in a period of time, but only qualitatively evaluate the overall performance of the bacterial flora, some characteristics in small sub-populations can be covered, and the researches are only qualitative ex-situ researches and cannot be quantitatively analyzed. Still other people adopt a spinning disk experiment to count the shear force applied when half of bacteria remain on the cyclone sheet, and the method belongs to qualitative ex-situ research, and can not accurately obtain the adhesion between bacteria and materials. In addition, the influence of the surface properties of materials on the adhesion properties of bacteria has been studied by students using tools such as a reflection interference contrast microscope, a micropipette, a membrane force probe, etc., but the overall performance of bacteria has been evaluated only semi-quantitatively, and the adhesion of bacteria cannot be quantitatively represented. Furthermore, bacteria themselves are highly heterogeneous, and studies of the bacteria as a whole may mask the individual's behaviour. Therefore, it is necessary to quantitatively study the adhesion behavior of individual bacteria on the surface of a material.

Means such as an optical tweezers method, a single-cell force spectrum technology, a nano indentation method, a cell separator method, a plasma diffraction imaging method and the like are developed at present to quantitatively study the adhesion force or the adhesion strength of single bacteria on the surface of a material. However, these methods all test the adhesion of bacteria in an ex-situ state, and during the testing process, the bacteria are in a dead state or an abnormal physiological metabolism state, which causes a great deviation between the result obtained by the testing and the actual situation, and the conclusion contrary to the fact is likely to be obtained. In addition, many bacteria have a close dynamic mass exchange (e.g., cytochromes, flavins) or electron transfer between the bacteria and the material when they adhere to the surface of the material. However, the current research means cannot realize quantitative detection of the actual adhesion behavior of single living bacteria on the surface of a material in the normal physiological metabolism process of the bacteria, and simultaneously obtain the mass exchange or electron transfer between the single living bacteria and the material.

Disclosure of Invention

In order to deeply explore the real adhesion behavior of bacteria on the surface of a material under the normal metabolism condition of the bacteria, the understanding and understanding of the formation mechanism of the biological film are further deepened, the effective control of the biological film is further realized, and a theoretical basis is finally provided for the design and development of related antibacterial and probiotic materials and equipment. The invention provides an in-situ test system and a test method for quantitatively detecting the adhesion behavior of single living bacteria, which can quantitatively detect the adhesion behavior of single living bacteria on the surface of a material (comprising the adhesion force of the bacteria and the material surface, the length of a biological macromolecule chain of an extramembranous protein, the average number of unfolding events and the average unfolding force) in the real physiological metabolism process of the bacteria, and simultaneously obtain an electronic transfer signal between the single living bacteria and the material.

The invention is realized by adopting the following technical scheme:

an in-situ test system for quantitatively detecting the adhesion behavior of single living bacteria comprises an electromagnetic shielding box, an electrochemical workstation, a computer, a damping table arranged in the electromagnetic shielding box, and an atomic force microscope and an optical inversion microscope which are arranged on the damping table; the atomic force microscope comprises an AFM base plate and an AFM scanner; the atomic force microscope is used for controlling the movement of the small ball probe and the sample stage, so that the grabbing and force curve testing of single living bacteria are realized. The optical inverted microscope is used for observing the bottom of the in-situ electrolytic cell from bottom to bottom, and determining the relative positions of bacteria and the small ball probe, so that the movement of the small ball probe and the sample stage is adjusted by matching with the atomic force microscope, and the grabbing and force curve testing of single living bacteria are realized;

the electromagnetic shielding box ensures good grounding and is used for eliminating electronic interference in the surrounding environment, in particular 50Hz power frequency interference. The atomic force microscope is used for controlling the movement of the small ball probe and the sample stage, so that the grabbing and force curve testing of single living bacteria are realized. The optical inverted microscope is used for observing the bottom of the in-situ electrolytic cell from bottom to determine the relative positions of bacteria and the small ball probe, so that the movement of the small ball probe and the sample stage is adjusted by matching with the atomic force microscope, and the grabbing and force curve testing of single living bacteria are realized. An environmental cavity is arranged on the objective table of the optical inverted microscope, the environmental cavity is a transparent quartz glass tubular body, and the upper edge of the environmental cavity is connected with an AFM base plate; the environment cavity is provided with an air inlet pipe and an air outlet pipe for adjusting the atmosphere in the environment cavity; the AFM scanner is fixedly arranged at the central round hole of the AFM base plate; a light source, an AFM optical microscope and a camera are arranged right above the AFM scanner and are used for observing the position of the small sphere probe and the laser position from top to bottom through the middle lens of the AFM scanner; a sample table is arranged in the environment cavity, and a round hole is formed in the sample table for observation by an optical inverted microscope; an in-situ electrolytic cell is arranged on the sample table, and living bacteria and bacterial culture solution are placed in the in-situ electrolytic cell; the sample table can move up and down, left and right and front and back; a probe is arranged in the AFM scanner, and a polydopamine layer is arranged on the outer surface of a small ball at the tip of the probe and used for grabbing single bacteria; the in-situ electrolytic cell is connected with an electrochemical workstation; the electrochemical workstation is used for applying different voltages to the working electrode in the in-situ electrolytic cell, controlling different testing conditions and detecting current signals between bacteria and the working electrode; the computer is used for setting experimental parameters and recording experimental data.

In the technical scheme, the in-situ electrolytic cell further comprises a bottom and a wall, wherein the bottom and the wall are sealed by O-shaped rings; the bottom of the tank is anode bonding glass, a working electrode and a counter electrode are arranged on the bottom of the tank, a reference electrode is arranged on the tank wall, and a vent pipe is also arranged on the tank wall. On the bottom of the tank, the counter electrode is arc-shaped, and the working electrode is positioned in the arc; and connecting the working electrode lead, the reference electrode lead and the counter electrode lead to corresponding wiring points of the in-situ electrolytic cell by using conductive silver paint, and connecting the leads to an electrochemical workstation.

Further, the counter electrode and the working electrode are obtained by evaporating a layer of titanium on the bottom surface of the tank and then evaporating a layer of gold on the titanium layer; the reference electrode is formed by plating a layer of silver chloride on the surface of a silver wire by adopting an electrochemical method, so that the part plated with the silver chloride layer accounts for about 4/5 of the length of the whole silver wire, then the silver wire plated with the silver chloride layer is placed in an in-situ electrolytic cell (fixed on the cell wall), the silver wire plated with the silver chloride layer is ensured to be in the cell, and the exposed silver wire is outside the cell and is used as a reference electrode wiring point.

Further, annular silica gel pads are arranged between the upper edge of the environmental cavity and the AFM base plate and between the lower edge of the environmental cavity and the objective table of the optical inverted microscope, so that a sealing environment is formed inside the environmental cavity and is used for maintaining different testing environments. The central circular hole of the optical inverted microscope stage is sealed by a cover glass.

Further, the sample stage may be moved up and down, left and right, and front and rear, specifically: a X, Y direction adjusting connecting rod and a magnetic lifting connecting rod are arranged below the AFM base plate, and a X, Y direction positioning hole for inserting the X, Y direction adjusting connecting rod is formed in the sample table; the sample platform is made of stainless steel, and the lifting connecting rod with magnetism can absorb the upper surface of the stainless steel sample platform so as to fix the sample platform.

Further, the damping table is suspended in the electromagnetic shielding box through four elastic bands and is used for eliminating the influence of vibration of the ground and the table top on the testing process. The inner surface of the shielding box is stuck with a sound absorbing material for eliminating the influence of environmental noise on the testing process.

The invention also provides an in-situ test method for quantitatively detecting the adhesion behavior of single living bacteria, which is realized based on the test system and comprises the following specific steps:

the reagents and vessels used in the following experimental procedure were sterilized in advance.

Firstly, 400 mu L of bacterial () suspension with an exponential growth phase is inoculated into a centrifuge tube containing 50mL of M9 solution, cultured for 24 hours at 30 ℃ and shakenThe rotation speed of the bed is 200rpm, and the bed grows to the concentration of OD _600nm ≈0.6-0.8。

Then, centrifugation was performed at 5000rpm for 5min, the supernatant was removed, then 50mL of PBS buffer was added to the centrifuge tube, resuspended, and centrifugation was performed at 5000rpm for 5min, and this step was repeated three times to obtain a suspension of bacteria in PBS.

The assembled in-situ electrolytic cell was ultrasonically cleaned in ultra-pure water for 15min, then dried in a nitrogen stream for 15min, and finally cleaned in an ultraviolet-ozone cleaner for 15min.

10 mu L of bacteria are dripped on glass between an in-situ electrolysis Chi Zhonggong electrode and a counter electrode, and the bacteria liquid is not required to contact the two electrodes, particularly the working electrode, so that the surface of the electrode is prevented from being polluted by bacteria, and the authenticity and the sensitivity of the testing process are prevented from being influenced.

Then, the added bacterial liquid is allowed to stand on the glass between the working electrode and the counter electrode for 30min, so that bacteria are settled and adhered to the surface of the glass.

The in situ cell was tilted 30 degrees, the bottom of the in situ cell was rinsed with PBS buffer from top to bottom, with the counter electrode down and the working electrode up to prevent bacteria from adhering to the working electrode surface, the suspended bacteria were removed by this method, rinsing was repeated several times, and microscopy was used until the bacteria that settled and adhered to the glass surface were independently dispersed.

Then, the bacterial broth is added to the in situ cell and the in situ cell is mounted to the AFM sample stage, which is then mounted into the environmental chamber.

And (3) introducing corresponding gas from a gas inlet pipe on the right side of the environment cavity, paying attention to the three-way valve, controlling the gas flow to the in-situ electrolytic cell, avoiding violent bubbling disturbance so as to prevent bacteria from being disturbed from the surface of glass, closing the valve to a branch pipe of the in-situ electrolytic cell after 1h, stopping blowing gas to the in-situ electrolytic cell, and continuing blowing gas to the environment cavity so as to prevent air from entering.

Carefully remove an AFM pellet probe with forceps having triangular microcantilevers with a 2.5 μm diameter SiO at the tips of the microcantilevers ₂ Ball with ball shapeThe probe was put into an ultraviolet-ozone washer and washed for 10min. Then 0.4g dopamine hydrochloride powder was dissolved in 100mL Trizma buffer, which was then adjusted to pH=8.5 with 1mol/L NaOH solution.

The probe washed with the ultraviolet-ozone washer was placed in a clean petri dish, and 10mL of dopamine hydrochloride solution at ph=8.5 was added to the petri dish, and the probe was ensured to be completely immersed. The mixture was placed on a biological shaker for 15min at a shaker speed of 60rpm.

The probe was then removed from the dish with forceps, at which time dopamine hydrochloride had been completed on SiO ₂ And polymerizing the surfaces of the pellets to generate polydopamine, wherein the polydopamine layer is used as an adhesive for subsequent grabbing bacteria. The treated probe was carefully rinsed 3-5 times with ultra pure water to remove the remaining dopamine hydrochloride and salt ions on the surface.

And drying the cleaned probe in nitrogen flow for 30min for standby.

And opening an AFM controller, opening AFM control software, opening a light source and a camera, setting an AFM to enter a contact mode, controlling a stepping motor on an AFM base plate by using an Open switch on an AFM electronic box, and reducing the position of a sample stage to ensure that a probe is not contacted with a liquid level after the sample stage is installed into a scanner.

The dried probe was mounted in an AFM scanner, which was then mounted in the central circular hole of the base plate, and the scanner locking knob was screwed.

And then, a Close switch on the AFM electronic box is used for controlling a stepping motor to slowly lift the sample stage, and naked eyes observe the sample stage until the probe is completely immersed below the liquid level, and stopping lifting.

The Focus knob on the AFM optical microscope is adjusted to complete the focusing of the micro-cantilever.

Turning on an AFM laser switch, rotating a Y-direction knob (for controlling the Y-direction movement of laser) at the upper part of a scanner, and simultaneously observing that a laser spot in an image display panel of an optical microscope moves up and down to the vicinity of a micro-cantilever, but cannot touch the micro-cantilever, wherein the specific adjusting position is ensured to touch the micro-cantilever when the laser moves left and right.

Turning an X-direction knob (for controlling the X-direction movement of the laser) at the upper part of the scanner, and simultaneously observing an image display panel of the optical microscope to enable the laser spot to move towards the micro-cantilever; then, the change of the laser in the white panel on the scanner hardware is closely watched, when the laser hits the edge of one side of the micro-cantilever, the elongation phenomenon occurs, and the knob is continuously rotated until the laser hits the edge of the other side of the micro-cantilever, at the moment, the elongation phenomenon occurs; and then rotating the knob in the opposite direction to perform micro-adjustment, and observing that the two sides of the laser on the white panel are not elongated, wherein the laser is positioned on the center line of the micro-cantilever.

Rotating a Y-direction knob at the upper part of the scanner, and simultaneously observing and observing an optical microscope image display panel to enable a laser spot to move towards the tip direction of the micro-cantilever; then, the laser changes in the white panel on the scanner hardware are closely watched, and at the moment, the laser reflection point intensity is gradually weakened until the laser reflection point disappears; and then the Y-direction knob is reversed, the laser is slowly appeared in the white panel from weak to strong until the laser is brightest (the Y-direction knob can not be continuously rotated again, so that the laser is not positioned at the tip of the micro-cantilever, and the detection sensitivity is reduced), and the laser is positioned at the tip of the micro-cantilever.

The method is characterized in that a Close switch on an electronic box is utilized to control a stepping motor on an AFM base plate to drive a lifting connecting rod with magnetism to move upwards, a sample table is initially lifted, a Focus knob is turned at moment to observe and shorten the distance between the tip of a micro-cantilever and the bottom of a pool, the bottom of the pool is prevented from contacting the tip of the micro-cantilever, and the bottom of an in-situ electrolytic cell is initially Close to the tip of the micro-cantilever by the method, so that the subsequent automatic approaching time is shortened.

The photodetector is installed in the scanner, the nut on the left side and the lower side of the detector is rotated to maximize SUM value (which is more than 1V) on the laser alignment panel, meanwhile, the reflection value is between-2V and-1.5V, the reflection value is as close to 0V as possible (which is within +/-0.2V), and the fluctuation range of the reflection value and the reflection value can be larger at the moment, because the probe micro-cantilever enters the liquid medium from the gas medium, the internal stress is larger, and the fluctuation of the micro-cantilever is larger.

And standing for a period of time (about 1 h) to release the internal stress of the probe micro-cantilever, readjusting the position of the detector window to ensure that the reflection value is between-2 and-1.5V, wherein the Friction value is as close to 0V as possible (within +/-0.2V), and performing the next operation after the reflection value and the Friction value are kept stable.

Under the assistance of a camera, the position of a working electrode in an image display panel of the optical microscope is observed, a X, Y-direction sample stage on an AFM base plate is adjusted to move and adjust a micrometer, and the sample stage is moved in the horizontal direction until the working electrode is right below a probe micro-cantilever.

Then, in the contact mode, setting the setpoint value to be 0-0.5, setting the stop at value to be 0, clicking an Approxh button at the speed of 0.5 mu m/s, so that the in-situ electrolytic cell slowly rises, and after the system automatically stops, the small ball at the tip of the probe just contacts the surface of the working electrode.

Then, the Withdraw value was set to 20 μm, and the Withdraw button was clicked to lower the sample stage by 20 μm, at which point the probe tip ball was 20 μm from the working electrode surface. Observing the bottom of the in-situ electrolytic cell by using an optical inversion microscope, slowly adjusting the focal length of the optical inversion microscope, and moving the sample stage in the X, Y direction on the AFM base plate in a matching manner to adjust the micrometer, so that the sample stage moves in the horizontal direction until bacteria and a small ball of the probe tip between the working electrode and the counter electrode are seen in an eyepiece of the optical inversion microscope.

The micrometer is continually fine-tuned so that a single bacterium is located just under the probe tip pellet. Then, in the contact mode, setting the setpoint value to be 0-0.5, the stop at value to be 0, the loading force value to be 0.5nN, and clicking an Apprach button at the speed of the stepping motor of 0.5 mu m/s to enable the sample stage to slowly lift, and after the system is automatically stopped, the small ball at the tip of the probe just contacts a single bacterium.

The probe tip pellet is held in contact with the bacteria for at least 5 minutes, and the polydopamine layer wrapped on the outer surface of the pellet acts as an adhesive to fix the bacteria on the pellet. Then, the Withdraw value was set to 20. Mu.m, the Withdraw button was clicked, the in situ cell was lowered by 20. Mu.m, and the pellet probe picked up the individual bacteria from the glass surface.

And then, under the assistance of a camera, observing the position of a working electrode in an image display panel of the optical inverted microscope, and adjusting the X, Y-direction sample stage on the AFM base plate to move and adjust the micrometer, so that the sample stage moves in the horizontal direction until the working electrode is right below the probe micro-cantilever.

And connecting the working electrode lead, the reference electrode lead and the counter electrode lead to corresponding wiring points of the in-situ electrolytic cell by utilizing conductive silver paint, detecting whether the connection is normal or not by using a universal meter before testing, eliminating the possibility of short circuit and open circuit, and then connecting the leads to an electrochemical workstation.

Closing the electromagnetic shielding box door.

Starting an electrochemical workstation, setting timing current testing parameters in the electrochemical workstation software, applying voltage 0V vs. Ag/AgCl, running for 30min, and clicking for running at data intervals of 20 s.

Then, in the contact mode, setting the setpoint value to be 0-0.5, the stop at value to be 0, the loading force value to be 0.5nN, and clicking an Apprach button at the speed of the stepping motor of 0.5 mu m/s to enable the sample table to slowly rise, and after the system is automatically stopped, the bacteria are contacted with the working electrode at the moment.

Then click open Force vs Distance function box, set micro-cantilever motion speed Rate value to 0.2 μm/s, force distance value to 1 μm, loading force value to 0.5nN,Data points value to 3000,Holding Time value to 5s. Clicking the Start button can Start the force curve test.

And carrying out quantitative analysis and numerical simulation on the force curve obtained by the test to obtain the maximum adhesion force between bacteria and the electrode surface, the length of the extracellular protein biological macromolecular chain, the average unfolding event times and the average unfolding force.

In addition, to obtain bacterial and electrode surface current signals, the following experiments were performed:

the Withdraw value was set to 20 μm and the Withdraw button was clicked to drop the in situ cell by 20 μm.

Then, in the contact mode, setting the setpoint value to be 0-0.5, the stop at value to be 0, the loading force value to be 0.5nN, and clicking an Apprach button at the speed of the stepping motor of 0.5 mu m/s to enable the in-situ electrolytic cell to slowly lift, and after the system is automatically stopped, contacting bacteria with a working electrode at the moment.

The timing current test parameters are set in the electrochemical workstation software, the applied voltage is 0V vs. Ag/AgCl, the running time is 30min, and the data interval is 20s.

Clicking to run, and starting to perform timing current test to obtain timing current curves of bacteria and electrode surfaces.

After the experiment, the living state of the bacteria immobilized on the probe tip was checked by using a bacteria living and dead identification box (L7012 live/dead BacLight bacterial viabilitykits), 7.5. Mu.L of the stain component A and 7.5. Mu.L of the stain component B were added to a centrifuge tube and mixed uniformly, then 5mL of PBS buffer was added to the centrifuge tube, mixed uniformly again, and poured into a clean petri dish.

Carefully taking off the small ball probe from the scanner by using forceps, immersing the small ball probe in a culture dish, wrapping the culture dish by using double-layer tin foil paper, and standing for 15min in a dark place.

Then, the small ball probe is taken out by forceps, the small ball probe is observed under a fluorescence microscope, the excitation light wavelength is 485nm, if the bacteria emit green fluorescence, the bacteria are proved to survive in the test process, and the experimental data are reliable.

The beneficial effects of the invention are as follows:

the test system of the present invention is used to test the micro-cantilever tip SiO by grabbing a single bacterium onto the micro-cantilever tip SiO ₂ On the pellets, the study on the adhesion behavior between single bacteria and materials can be realized, so that the problem that the characteristics of small sub-populations and bacterial individuals are covered when the whole bacterial population is studied by the traditional method is solved.

The invention can realize the in-situ characterization of the real adhesion behavior of single bacteria on the surface of a material under the normal metabolism condition of the bacteria, and provides an in-situ electrolytic cell which can provide normal metabolism conditions for the bacteria in the test process, such as liquid environment, nutrient substances, aerobic and anaerobic conditions, temperature and electron acceptors (electrodes or other chemical components), and can solve the problem that the bacteria need to be killed or abnormal survival conditions are provided for the bacteria in the traditional research method by matching with the test of the survival state of the bacteria after the experiment, thereby ensuring the authenticity and the reliability of the test result.

The invention can realize quantitative characterization of the actual adhesion behavior of single bacteria on the surface of the material. The specific value of the adhesion force between the bacteria and the working electrode, the length of the bacterial membrane outer protein biological macromolecular chain, the number of unfolding events and the size of the unfolding force can be accurately obtained through quantitative analysis and numerical simulation calculation of the force curve, and the electronic transmission information between the bacteria and the materials can be obtained at the same time. The quantitative adhesion behavior of bacteria on the surface of the material and the electronic transmission information between the bacteria and the material are subjected to correlation analysis, so that the method can be used for deep exploration of a bacterial adhesion mechanism and a biological film formation mechanism, further effective control of the biological film is realized, and finally a theoretical basis is provided for design and development of related antibacterial and probiotic materials and equipment.

The invention has simple structure and strong openness, and can research the adhesion behavior of various bacteria, cells and molecules on the surfaces of various materials (such as metal, alloy, polymer, semiconductor, ceramic and the like) according to actual demands. The invention provides an environment cavity to realize the control of test atmosphere such as oxygen, anaerobic and CO ₂ 、CH ₄ And the like. In addition, the invention provides an in-situ electrolytic cell, which can apply different electrode potentials to materials and add bacterial culture solutions with different components into the in-situ electrolytic cell, so that the adhesion behavior of bacteria can be explored in different electrochemical environments, and the actual requirements of different research fields can be met.

Drawings

FIG. 1 is an in situ test system for quantitatively detecting the adhesion behavior of a single living bacterium in accordance with the present invention;

FIG. 2 is a core test portion of the in situ test system;

FIG. 3 is an AFM sample stage;

FIG. 4 is a plot of in situ cell floor dimensions in mm;

FIG. 5 is an in situ cell assembly flow diagram;

FIG. 6 is a view showing bacteria dropped between a working electrode and a counter electrode;

FIGS. 7 (a), 7 (b), 7 (c) are single bacteria grasping procedures; 7 (d), 7 (e), 7 (f) are adhesion behavior test flows;

FIG. 8 (a) is a force profile test result obtained with a probe with bacteria at the tip; 9 (b) is a force curve test result obtained when bacteria fall off;

FIG. 9 is a worm chain model fit;

fig. 10 (a) is the maximum adhesion; 11 (b) is the extracellular protein biomacromolecule chain length; 11 (c) is the average number of events of the extracellular protein biomacromolecule chains; 11 (d) is the average expansion force of the biological macromolecular chains of the extramembranous proteins;

FIG. 11 is a diagram showing the metabolic process of Kaschin-situ test system operation principle;

FIG. 12 is a graph of the timed current for bacteria and electrode surfaces at different electrode potentials;

wherein, 1, a gas cylinder; 2. a core test section; 3. a shock absorbing platform; 4. an elastic band; 5. an electromagnetic shielding box; 6. a working electrode lead; 7. a reference electrode lead; 8. a counter electrode lead; 9. an electrochemical workstation; 10. an AFM controller; 11. a computer host; 12. a computer display; 13. a ground wire; 14. a test bed; 15. an eyepiece; 16. an optical inverted microscope; 17. a camera; 18. a light source; 19. AFM optical microscope; 20. an AFM scanner; 21. an AFM base plate; 22. an optical inverted microscope objective; 23. moving the sample stage in the X direction to adjust the micrometer; 24. an X-direction adjusting link 25, a lifting link with magnetism; 26. a stepping motor; 27. a Y-direction adjusting connecting rod; 28. moving the sample stage in the Y direction to adjust the micrometer; 29. an environmental chamber; 30. a sample stage; 31. an objective table; 32. an in situ electrolytic cell; 33. a small ball probe; 34. a cover slip; 35. an air outlet pipe; 36. an air inlet pipe; 37. an X-direction knob; 38. a Y-direction knob; 39. positioning holes in the X direction; 40. positioning holes in the Y direction; 41. inverting the microscope viewing aperture; 42. anodic bonding glass; 43. a counter electrode; 44. a working electrode; 45. a reference electrode; 46. a vent pipe; 47. a pool wall; 48. a screw; 49. an O-ring; 50. a counter electrode lead wire connection point; 51. working electrode lead wire connection points; 52. a reference electrode lead wire junction; 53. a dispersed bacteria; 54. a micro-cantilever; 55. SiO (SiO) ₂ A pellet; 56. bacterial cultureA nutrient solution; 57. single osnescentella sp.

Detailed Description

An in-situ test system for quantitatively detecting the adhesion behavior of single living bacteria, as shown in fig. 1, comprises an electromagnetic shielding box 5, an electrochemical workstation 9, a computer (comprising a computer host 11 and a computer display 12), a shock absorption table 3 arranged in the electromagnetic shielding box 5, and an atomic force microscope and an optical inversion microscope 16 arranged on the shock absorption table 3; the atomic force microscope comprises an AFM base plate 21 and an AFM scanner 20; the computer is used for setting experimental parameters and recording experimental data.

The core test portion 2 of the in situ test system of the present invention is shown in fig. 2 as being composed of an atomic force microscope, an optical inverted microscope 16, an in situ cell 32, and an environmental chamber 29. A transparent quartz glass tube body having an outer diameter of 135mm, a wall thickness of 5mm and a height of 100mm was mounted on the stage 31 of the optical inversion microscope as the environmental chamber 29, an annular silica gel pad was mounted between the lower edge of the environmental chamber 29 and the stage 31 of the optical inversion microscope, the center circular hole of the stage 31 of the optical inversion microscope was sealed with the cover glass 34, an annular silica gel pad was also mounted between the upper edge of the environmental chamber 29 and the AFM base plate 21, and the AFM scanner 20 was fixedly mounted to the center circular hole of the AFM base plate 21 (the scanner locking knob was screwed to form a sealed environmental chamber for maintaining different test environments). An air inlet pipe 36 (the air inlet pipe 36 is connected with the air bottle 1) and an air outlet pipe 35 are arranged on the right side of the environment cavity 29 and used for adjusting the air atmosphere in the environment cavity 29. The environment chamber 29 is internally provided with a sample stage 30, as shown in fig. 3, which has a diameter of 80mm and a thickness of 3mm, is made of stainless steel (can be sucked by the lifting connecting rod 25 with magnetism), and is provided with a round hole with a diameter of 30mm in the middle as an observation hole 41 of an inverted microscope for observation of the inverted microscope, and is provided with an AFM sample stage X-direction positioning hole 39 and an AFM sample stage Y-direction positioning hole 40 for being matched with the

lifting connecting rods

24 and 27 with a X, Y direction so as to adjust the position of the sample stage 30 in the horizontal direction. Four threaded holes are provided around the sample stage 30 for securing the in situ cell 32.

The in-situ electrolytic cell 32 comprises a cell bottom and a cell wall 47, wherein the cell bottom and the cell wall 47 are sealed by an O-shaped ring 49; the shape and the size of the pool bottom are shown in fig. 4, the pool bottom is obtained by evaporating a layer of titanium and then evaporating a layer of gold on the anode bonding glass 42 by adopting a vacuum evaporation method, the size is 45mm, the width is 45mm, the thickness is 0.5mm, and the surface roughness is less than 10nm. According to the shape and size shown by the hatched portion in fig. 4, a layer of titanium having a thickness of 50nm was first deposited on the upper surface of the bottom of the tank by vacuum deposition as a buffer layer and an adhesive layer. Then, a layer of gold with a thickness of 300nm was evaporated on top of the titanium layer. Finally, a pool bottom as shown in fig. 5 (a) is formed in which a small-area hatched gold plating layer is used as the working electrode 44 and a large-area hatched gold plating layer is used as the counter electrode 43. Round holes are formed at four corners of the pool bottom for fixing the pool wall 47. The wall 47 is made of polytetrafluoroethylene, the wall 47, the bottom and the sample table 30 are fixed together by four screws 48, and the wall 47 and the bottom are sealed by a silica gel O-ring 49 to prevent the leakage of the solution, as shown in FIG. 5 (b). A vent 46 (for regulating the dissolved gas content of the solution in the in situ cell 32) is also provided on the wall 47. The reference electrode 45 is fixed on the tank wall 47, the reference electrode 45 is a silver wire plated with silver chloride, as shown in fig. 5 (a, b), which is obtained by a chronopotentiometric method, firstly, a silver wire with the diameter of 0.5mm is washed clean by a nitric acid bubble, then is inserted into a beaker containing 0.1mol/L KCl solution, the silver wire is a working electrode, the platinum wire is a counter electrode, a saturated calomel electrode is a reference electrode, a constant current parameter of 1mA is set, the running time is 15min, and finally the obtained silver chloride plating layer is purplish brown, as shown in fig. 5 (a). Note that the silver wires were inserted to about 4/5 length so that the silver chloride coated portion was 4/5 of the length of the entire silver wire, and then the silver chloride coated silver wires were placed in the in situ electrolytic cell 32, ensuring that the silver chloride coated silver wires were all the portion inside the cell, with the bare silver wires outside the cell, as shown in fig. 5 (b). Finally assembled into an in situ cell 32 as shown in fig. 5 (c). The wall 47 of the in situ cell 32 is slightly smaller than the bottom of the cell, leaving a working electrode lead junction 51, a reference electrode lead junction 52 and a counter electrode lead junction 50 around, as shown in fig. 5 (d).

As shown in fig. 2, the in-situ cell 32 assembled on the sample stage 30 (fig. 5 (c)) is mounted under the AFM base plate 21 such that the X, Y

direction adjusting links

24, 27 under the AFM base plate 21 are inserted into X, Y direction positioning holes 39, 40, respectively, on the sample stage 30, while the magnetically attached lifting link 25 is made to attract the upper surface of the sample stage 30 made of stainless steel. The sample stage 30 and the in-situ electrolytic cell 32 can be moved in the horizontal direction by adjusting the movement of the sample stage in X, Y direction and the sample stage 30 and the in-situ electrolytic cell 32 can be moved in the vertical direction by controlling the stepping motor 26 to drive the magnetic lifting connecting rod 25. The position of the sample stage 30 is adjusted so that the optical inverted microscope objective 22, the inverted microscope viewing aperture 41, the in situ cell 32, the ball probe 33, the AFM scanner 20, the camera 17, the light source 18, and the AFM optical microscope 19 are collinear.

As shown in fig. 1, in order to reduce the influence of disturbance of the test stand 14 on the test process and to improve the test sensitivity, the core test part 2 shown in fig. 2 is placed on the damper stand 3, and the damper stand 3 is suspended in the electromagnetic shielding box 5 by four elastic bands 4. The electromagnetic shielding box 5 is well connected with the ground through the grounding wire 13, so that electronic interference, especially power frequency interference of 50Hz, can be effectively eliminated. The inner surface of the electromagnetic shielding box 5 is stuck with a sound absorbing material (such as a sound absorbing sponge), so that the influence of environmental noise on the testing process can be eliminated.

As shown in fig. 2, the working electrode lead 6, the reference electrode lead 7 and the counter electrode lead 8 were connected to the corresponding connection points of the in-situ electrolytic cell 32 (fig. 5 (d)) using conductive silver paint, and the connection was checked for normality by using a multimeter before the test, excluding the possibility of short circuits and open circuits, and then the leads were connected to the electrochemical workstation 9. The gas enters the environment cavity through the pressure reducing valve and the gas flow controller.

The following takes a conventional gram-negative bacterium, namely, kanesia (S.oneidensis MR-1), as an example of the adhesion behavior study on the gold electrode surface, and the operation steps and precautions of the in-situ test system of the present invention will be described in detail with reference to the accompanying drawings. The technical solution of the present invention will be further described with reference to the accompanying drawings and specific examples, which are given by way of illustration of the preferred examples of the method and principle of the present invention, and are not meant to be limiting in any way.

The bacteria are electrochemically active bacteria, and are model species used to study the interactions between bacteria and the surface of materials. The Ornidazole bacteria can utilize relevant biological enzymes in the body to decompose some organic molecules (such as lactic acid, acetic acid and the like) into protons, carbon dioxide and the like, and simultaneously obtain electrons, a small part of the electrons are used for the growth and propagation of the bacteria, and the rest of the electrons are transferred to an electron acceptor in vitro through extracellular electron transfer, and the electron acceptor can be an electrode, ore, oxygen, riboflavin and the like. Namely, the oxidation reaction of organic molecules in bacteria is equivalent to the metabolism of osnescentella as shown in fig. 11. Therefore, the bacteria are often used in microbial fuel cells to treat sewage using a biofilm formed by the bacteria growing on the surface of an electrode material while generating electric power.

The bacteria are hereinafter referred to as "bacteria". Before the experiment, the bacteria were transferred from-80 ℃ refrigerator to-20 ℃ refrigerator and left for 2h. Then, the bacteria were transferred from the-20℃refrigerator to the 0℃refrigerator and left for 2 hours. 400. Mu.L of the bacterial liquid was pipetted into a centrifuge tube containing 20mL of LB liquid medium.

Culturing at 30deg.C until the bacterial index increases (OD _600nm Approximately 1.0-1.2) the rotation speed of the biological shaking table is 200rpm. Then, 400. Mu.L of bacteria (OD _600nm About 1.0-1.2) the suspension was inoculated into a centrifuge tube containing 50mLM solution, incubated at 30℃for 24h, shaking at 200rpm, OD _600nm ≈0.6-0.8。

Then, centrifugation was performed at 5000rpm for 5min, the supernatant was removed, then 50ml of buffer was added to the centrifuge tube, resuspended, and centrifugation was performed at 5000rpm for 5min, and this step was repeated three times to obtain a suspension of bacteria in PBS.

The bottom of the tank as shown in FIG. 5 (a) was ultrasonically cleaned in ultra-pure water for 15min, then dried in a nitrogen gas stream for 15min, and finally cleaned in an ultraviolet-ozone cleaner for 15min.

Then 10. Mu.L of the bacterial liquid was dropped onto the glass between the working electrode 44 and the counter electrode 43 on the bottom of the cell as shown in FIG. 5 (a), taking care not to contact the bacterial liquid with both electrodes, especially taking care not to contact the working electrode 44, preventing bacteria from contaminating the electrode surface, affecting the authenticity and sensitivity of the test procedure. Then, the added bacterial liquid was allowed to stand on the glass between the working electrode 44 and the counter electrode 43 for 30 minutes, so that the bacteria were settled and adhered to the surface of the anodic bonding glass 42.

Then, the bottom of the cell was inclined at 30 degrees, washed with PBS buffer from top to bottom, taking care that the counter electrode 43 was below and the working electrode 44 was above to prevent bacteria from adhering to the surface of the working electrode 44, the suspended bacteria were removed by this method, washing was repeated several times, and observation was performed with a microscope until the bacteria settled and adhered to the surface of the anodic bonding glass 42 were independently dispersed, to obtain dispersed bacteria 53, as shown in FIG. 6.

The bottom of the cell was then quickly assembled with O-ring 49, wall 47, and sample stage 30 by four screws 48 as shown in FIG. 5 (b), to form the in situ cell 32 as shown in FIG. 5 (c), 2.5mL of bacterial broth (containing 18mmol/L sodium lactate) was added to the in situ cell 32 as the organic material required for bacterial metabolism, and then the sample stage 30 and in situ cell 32 were mounted in the environmental chamber 29 as shown in FIG. 2.

As shown in fig. 2, nitrogen is introduced from the right air inlet 36 of the environmental chamber 29, oxygen in the solution in the environmental chamber 29 and in-situ electrolytic cell 32 is removed under the action of the tee joint in the environmental chamber, the tee joint valve is adjusted, the air flow to the in-situ electrolytic cell 32 is controlled, severe bubbling disturbance is avoided, bacteria are prevented from being disturbed from the surface of the anode bonding glass 42, after 1h, the valve to the branch pipe of the in-situ electrolytic cell is closed, the air blowing to the in-situ electrolytic cell 32 is stopped, and the air blowing to the environmental chamber 29 is continued, so that the air is prevented.

An AFM pellet probe 33 (k=0.06N/m) having a triangular micro-cantilever 54 with a SiO 2.5 μm diameter at the tip of the micro-cantilever 54 was carefully removed with forceps ₂ The pellets 55 were put into an ultraviolet-ozone washer to wash 10min. Then 0.4g dopamine hydrochloride powder was dissolved in 100mL Trizma buffer, which was then adjusted to pH=8.5 with 1mol/L NaOH solution.

The pellet probe 33 washed with the ultraviolet-ozone washer described above was placed in a clean petri dish, and 10mL of dopamine hydrochloride solution at ph=8.5 was added to the petri dish, and the pellet probe 33 was ensured to be completely immersed. The mixture was placed on a biological shaker for 15min at a shaker speed of 60rpm.

The pellet probe 33 was removed from the petri dish with forceps and the dopamine hydrochloride had been completed on the SiO ₂ The surface of the pellet 55 polymerizes to form polydopamine, which acts as a binder to modify bacteria on the pellet. The treated pellet probe 33 was carefully rinsed 3-5 times with ultra pure water to remove the remaining dopamine hydrochloride and salt ions on the surface.

The cleaned pellet probe 33 was then dried in a nitrogen stream for 30 minutes for use.

The AFM controller 10 is opened, AFM control software is opened, the camera 17 and the light source 18 are opened, the AFM is set to enter a contact mode, an Open switch on the electronic box is utilized to control the stepping motor 26 on the AFM base plate 21, the position of the sample table 30 is lowered, and the small ball probe 33 is ensured not to contact the liquid level after the sample table is installed into the scanner 20.

The dried ball probe 33 is installed into the AFM scanner 20, and then the AFM scanner 20 is installed into the central circular hole of the AFM base plate 21, and the scanner locking knob is screwed. Then, the stepping motor 26 is controlled by a Close switch on the electronic box, so that the sample stage 30 is slowly lifted, and the sample stage is observed visually until the small ball probe 33 is completely immersed below the liquid level, and the lifting is stopped.

Adjusting the Focus knob on AFM optical microscope 19 completes the focusing of microcantilever 54.

Turning on the AFM laser switch, turning the Y-direction knob 38 on the upper portion of the scanner while observing the movement of the laser spot up and down in the optical microscope image display panel to the vicinity of the micro-cantilever 54, but not touching the micro-cantilever 54, the specific adjustment position should be such that the laser beam touches the micro-cantilever 54 when moving left and right.

Turning the scanner upper X-direction knob 37 while observing the optical microscope image display panel, causing the laser spot to move toward the micro-cantilever 54; then, looking at the change of the laser in the white panel on the scanner hardware, when the laser hits the edge of one side of the micro-cantilever 54, the elongation phenomenon occurs, and the X-direction knob 37 is continuously rotated until the laser hits the edge of the other side of the micro-cantilever 54, at this time, the elongation phenomenon occurs; the X-direction knob 37 is then rotated in the opposite direction to make a fine adjustment while observing that the laser on the white panel is not elongated on either side, with the laser on the midline of the microcantilever 54.

Turning the scanner upper Y-direction knob 38 while viewing the optical microscope image display panel to move the laser spot toward the tip of the micro-cantilever 54; then, the laser changes in the white panel on the scanner hardware are closely watched, and at the moment, the laser reflection point intensity is gradually weakened until the laser reflection point disappears; the Y-direction knob 38 is reversed again, and the laser light is slowly appeared in the white panel from weak to strong, and is adjusted until the laser light is brightest (the Y-direction knob 38 can not be turned any more, so that the laser light is not positioned at the tip of the micro-cantilever 54, and the detection sensitivity is reduced), and the laser light is positioned at the tip of the micro-cantilever 54.

The step motor 26 is controlled by a Close switch on the electronic box to initially lift the sample stage 30, and the Focus knob is turned to observe and shorten the distance between the tip of the micro-cantilever 54 and the bottom of the cell, so as to prevent the bottom of the cell from contacting the tip of the micro-cantilever 54.

The photodetector is mounted in the scanner, the nuts on the left and below the detector are turned to maximize the SUM value on the laser alignment panel (which must be greater than 1V), while the reflection value is between-2 and-1.5V, and the reflection value is as close to 0V as possible (within + -0.2V), at which time the fluctuation range of the reflection value and the reflection value may be relatively large because the probe micro-cantilever 54 enters the liquid medium from the gaseous medium, and the internal stress is relatively large, so that the micro-cantilever 54 fluctuates greatly.

The sample is left for a period of time (about 1 h) to allow the internal stress of the micro-cantilever 54 of the ball probe 33 to be released, the position of the detector window is readjusted so that the reflection value is between-2 and-1.5V, the Friction value is as close to 0V as possible (within + -0.2V), and the next operation is performed after the reflection value and the Friction value are kept stable.

With the aid of the camera 17, the position of the working electrode 44 in the image display panel of the optical microscope is observed, the X, Y direction sample stage on the AFM base plate 21 is adjusted to move the adjusting micrometer 23, 28, and the in-situ electrolytic cell 32 is moved in the horizontal direction until the working electrode 44 is right below the micro-cantilever 54 of the ball probe 33.

Then, in the contact mode, setting the setpoint value to 0-0.5, the stop at value to 0, the speed of the stepper motor 26 to 0.5 μm/s, clicking the Appproach button to slowly lift the in-situ electrolytic cell 32, and after the system is automatically stopped, the tip SiO of the small ball probe 33 is at the same time ₂ The bead 55 is just touching the surface of the working electrode 44.

Then, the Withdraw value was set to 20 μm, and the Withdraw button was clicked to lower the in-situ cell 32 by 20 μm, at which time the tip of the ball probe 33 was SiO ₂ The beads 55 are 20 μm from the surface of the working electrode 44. As shown in FIG. 2, the bottom of the in situ cell 32 is observed with the optical inverted microscope 16, the focal length of the inverted microscope is slowly adjusted, and the micrometer 23, 28 is adjusted in coordination with the X, Y direction sample stage movement on the AFM base plate 21, so that the in situ cell 32 is moved in the horizontal direction until the SiO at the tip of the bacteria and pellet probe 33 between the working electrode 44 and counter electrode 43 is seen in the eyepiece 15 of the optical inverted microscope 16 ₂ A pellet 55.

As shown in FIG. 7 (a), the movement of the sample stage in the direction of X, Y is continued to be finely adjusted to adjust the micrometer 23, 28 so that a single bacterium is just positioned at the tip SiO of the ball probe 33 ₂ Directly below the pellets 55. Then, in the contact mode, setting the setpoint value to 0-0.5, stop at 0, loading force 0.5nN, the speed of the stepping motor 26 is 0.5 μm/s, clicking an Apprach button to slowly lift the in-situ electrolytic cell 32, and after the system is automatically stopped, the tip of the ball probe 33 is SiO at the moment ₂ The pellet 55 is just touching a single bacterium as shown in fig. 7 (b).

Maintaining the tip SiO of the ball probe 33 ₂ The pellet 55 is contacted with bacteria for 5min, and the pellet is wrapped with polymerThe dopamine layer is used as a binder to fix bacteria on SiO ₂ On the pellets 55. After 5min, the Withdraw value was set to 20 μm, the Withdraw button was clicked, the in situ cell 32 was lowered by 20 μm, and the pellet probe 33 grasped individual bacteria from the surface of the anodic bonding glass 42, as shown in FIG. 7 (c).

Then, with the aid of the camera 17, the position of the working electrode 44 in the image display panel of the optical microscope is observed, the X, Y direction sample stage on the AFM base plate 21 is adjusted to move the adjusting micrometer 23, 28, and the in-situ electrolytic cell 32 is moved in the horizontal direction until the working electrode 44 is right below the micro cantilever 54 of the ball probe 33.

As shown in fig. 2, the working electrode lead 6, the reference electrode lead 7 and the counter electrode lead 8 were connected to the corresponding connection points of the in-situ electrolytic cell 32 (fig. 5 (d)) using conductive silver paint, and the connection was checked for normality by using a multimeter before the test, excluding the possibility of short circuits and open circuits, and then the leads were connected to the electrochemical workstation 9.

The electromagnetic shielding box 5 door is closed.

Starting the electrochemical workstation 9, setting timing current test parameters in the software of the electrochemical workstation 9, applying voltage of 0V vs. Ag/AgCl, running for 30min, performing data interval of 20s, and performing clicking operation.

Then, in the contact mode, set the setpoint value to 0-0.5, stop at 0, loading force 0.5nN, the stepper motor 26 speed 0.5 μm/s, click the Apprach button to slowly raise the in situ cell 32, and after the system has stopped automatically, the bacteria come into contact with the working electrode 44.

Then click open Force vs Distance function box, set the speed Rate of motion of micro-cantilever 54 to 0.2 μm/s, force distance to 1 μm, loading force to 0.5nN,Data points to 3000,Holding Time to 5s. Then click the Start button to begin the force profile test.

The force curve test results obtained are shown in fig. 8 (a). In fig. 8 (a), the dotted line represents the process in which bacteria gradually approach the surface of the working electrode 44, and the dotted line is kept horizontal from the point a until the bacteria contact the surface of the working electrode 44, and the state of the micro-cantilever 54 is shown in fig. 7 (d). At point b, the bacteria contact the surface of the working electrode 44, the dotted line is bent upward, and at point c, the set value of 0.5nN is reached, and the state is maintained for 5s, and the state of the micro-cantilever 54 is shown in FIG. 7 (e). The solid black line represents the gradual pulling of bacteria away from the surface of working electrode 44, starting at point d and straightening of microcantilever 54 at point e. From point e to point f, the micro-cantilever 54 slowly pulls the bacteria from the surface of the working electrode 44, and the state of the micro-cantilever 54 is shown in fig. 7 (f), and by calculating the absolute value of the ordinate of point f, the quantitative value of the maximum adhesion force between the bacteria and the surface of the working electrode 44 can be obtained. From point f, the bacterial body leaves the surface of working electrode 44 and the f-to-g force curve becomes serrated, which represents the process of gradual development of some extramembranous protein bio-macromolecular chains on the bacterial surface. At point g, the bio-macromolecular chains are out of contact with the surface of working electrode 44, and the force curve jumps from point g to point h. The average unfolding force of the extracellular protein biological macromolecular chains can be obtained by calculating the average value of the longitudinal coordinates of all the sawtooth-shaped part wave troughs, and the average unfolding event times of the extracellular protein biological macromolecular chains can be obtained by calculating the average value of the sawtooth number. Numerical simulation calculations were performed according to the worm chain model below, as shown in fig. 9, to obtain the average length of the extramembranous protein biomacromolecule chain.

Wherein F is the magnitude of the spreading force, z is the distance between the bacteria and the electrode surface, k _B Is Boltzmann constant, T is the test temperature, L _c To be straightened, to represent the length of the extracellular protein biomacromolecule chain, L _P To describe the flexibility of the extracellular protein biomacromolecule chains.

If bacteria fall off during the test, the obtained force curve test results are shown in FIG. 8 (b), and the absolute value of the ordinate of the f point in FIG. 8 (b) is the tip SiO of the ball probe 33 ₂ Maximum adhesion of the pellet 55 to the surface of the working electrode 44 due to SiO ₂ The pellets 55 are coated with a layer of polydopamine, thus SiO ₂ The pellet 55 and working electrode 44 surfaces also have some adhesion, however, the serrations willWill disappear due to SiO ₂ The surface of the beads 55 is free of macromolecular chains that can fold and unfold.

The above experiment was repeated by changing the electrode potential of the working electrode 44, collecting 100 to 200 force curves under each electrode potential condition, and carrying out statistical analysis on the force curves, and simultaneously carrying out significance analysis by adopting a double-tail unpaired t test, and obtaining the results of maximum adhesion force between bacteria and the surface of the working electrode 44, the length of the extracellular protein biological macromolecule chain, the average unfolding times and the average unfolding force under different electrode potentials, wherein the results are shown in fig. 10. As can be seen from fig. 10, as the electrode potential of the working electrode 44 increases, the adhesion between the bacteria and the electrode surface gradually increases, the length of the bacterial membrane outer protein macromolecular chain gradually shortens, and the average number of macromolecular chains and the average spreading force gradually increases. By significance analysis, if the calculated P <0.05, a significant difference can be considered between the two sets of data. If P <0.05, then there is considered to be an insignificant difference (NS) between the two sets of data.

In addition, in order to obtain a bacterial and working electrode 44 surface current signal, the following experiments were performed:

the in situ cell 32 is lowered by 20 μm by setting the Withdraw value to 20 μm and clicking the Withdraw button.

Clicking operation starts to perform a timing current test, the test principle is shown in fig. 11, a single oshneri bacterium 57 is metabolized in a bacterial culture solution 56, and some organic molecules (such as lactic acid, acetic acid and the like) can be decomposed into protons and carbon dioxide by utilizing relevant biological enzymes in the body, and electrons are obtained at the same time and transferred to a working electrode 44 through extracellular electronic transmission, so that the breathing process of the bacterium is completed. The electrode potential of the working electrode 44 was varied and the above experiment was repeated to obtain a timed current profile for bacteria and electrode surfaces as shown in fig. 12. It can be seen that as the electrode potential increases, the current between the bacteria and the electrode increases.

After the end of the experiment, the living state of the single Kaschin-Kaschin 57 immobilized at the tip of the pellet probe 33 was examined by using a bacteria live-dead identification box (L7012 live/dead BacLight bacterial viabilitykits), 7.5. Mu.L of the stain component A and 7.5. Mu.L of the stain component B were added to a centrifuge tube and mixed uniformly, then 5mL of PBS buffer was added to the centrifuge tube, and after mixing uniformly again, poured into a clean dish.

The pellet probe 33 was carefully removed from the AFM scanner 20 with forceps, and then the pellet probe 33 was immersed in the petri dish, wrapped with double-layered tin foil paper, and allowed to stand in a dark place for 15min.

Then, the pellet probe 33 was taken out with forceps, observed under a fluorescence microscope, and the excitation light wavelength was 485nm, if the bacteria emitted green fluorescence, it was confirmed that the bacteria survived the test procedure, and the experimental data was reliable.

Claims

1. An in-situ test method for quantitatively detecting the adhesion behavior of single living bacteria is characterized by being realized based on a test system for quantitatively detecting the adhesion behavior of single living bacteria, wherein the system comprises an electromagnetic shielding box, an electrochemical workstation, a computer, a damping table arranged in the electromagnetic shielding box, an atomic force microscope and an optical inversion microscope which are arranged on the damping table; the atomic force microscope AFM comprises an AFM base plate and an AFM scanner; the electromagnetic shielding box is grounded and used for eliminating electronic interference in the surrounding environment; the damping table is used for eliminating the influence of vibration of the ground and the table top on the test process;

An environmental cavity is arranged on the objective table of the optical inverted microscope, the environmental cavity is a transparent quartz glass tubular body, and the upper edge of the environmental cavity is connected with an AFM base plate; the environment cavity is provided with an air inlet pipe and an air outlet pipe for adjusting the atmosphere in the environment cavity; the AFM scanner is fixedly arranged at the central round hole of the AFM base plate; a light source, an AFM optical microscope and a camera are arranged right above the AFM scanner and are used for observing the position of the small sphere probe and the laser position from top to bottom through the middle lens of the AFM scanner; a sample table is arranged in the environment cavity, and a round hole is formed in the sample table for observation by an optical inverted microscope; an in-situ electrolytic cell is arranged on the sample table, and living bacteria and bacterial culture solution are placed in the in-situ electrolytic cell; the sample table can move up and down, left and right and front and back; a probe is arranged in the AFM scanner, and a polydopamine layer is arranged on the outer surface of a small ball at the tip of the probe and used for grabbing single bacteria; the in-situ electrolytic cell is connected with an electrochemical workstation; the electrochemical workstation is used for controlling different in-situ test conditions and detecting current signals between bacteria and working electrodes in the in-situ electrolytic cell; the optical inverted microscope is used for observing the bottom of the in-situ electrolytic cell from the bottom down and determining the relative positions of bacteria and the small ball probe, so that the movement of the small ball probe and the sample table is adjusted by matching with the atomic force microscope, and the grabbing and force curve testing of single living bacteria are realized; the computer is used for setting experimental parameters and recording experimental data;

The in-situ electrolytic cell comprises a cell bottom and a cell wall, wherein an O-shaped ring is adopted to seal the cell bottom and the cell wall; the bottom of the tank is anode bonding glass, a working electrode and a counter electrode are arranged on the bottom of the tank, a reference electrode is arranged on the wall of the tank, and a vent pipe is also arranged on the wall of the tank;

the test method comprises the following steps: under the assistance of an optical inverted microscope, controlling the front and back, left and right and up and down movement of a sample stage, then grabbing single living bacteria by using a probe arranged in an AFM scanner, and then continuously controlling the front and back, left and right and up and down movement of the sample stage, so that the probe grabbing bacteria is positioned right above a working electrode, and setting parameters of an atomic force microscope and an electrochemical workstation; clicking to run after setting parameters, so as to obtain a force curve and a timing current curve of bacteria and the electrode surface; in the test process, stopping blowing air into the in-situ electrolytic cell, and continuing blowing air into the environment cavity to prevent air from entering;

the maximum adhesion force between bacteria and the electrode surface, the length of the biological macromolecular chain of the outer membrane protein, the average unfolding event times and the average unfolding force can be obtained by carrying out quantitative analysis and numerical simulation on the force curve obtained by the test; the current signal between the bacteria and the electrode can be obtained through timing current curve;

Calculating the average value of the longitudinal coordinates of all the sawtooth-shaped part wave troughs in the force curve to obtain the average unfolding force of the extracellular protein biological macromolecular chain; calculating the average value of the number of saw teeth in the force curve to obtain the average number of spreading events of the extracellular protein biomacromolecule chains; performing numerical simulation calculation according to a worm chain model to obtain the average length of the extramembranous protein biological macromolecular chain, wherein the average length is specifically as follows:

wherein F is the magnitude of the unfolding force; z is the distance between the bacteria and the electrode surface; k (k) _B Is the boltzmann constant; t is the test temperature; l (L) _c For the straightened length, represents the length of the biological macromolecular chain of the extramembranous protein; l (L) _P To describe the flexibility of the extracellular protein biomacromolecule chains.

2. The in situ test method for quantitatively detecting adhesion behavior of a single living bacterium of claim 1, wherein on the bottom of the cell, the counter electrode is arc-shaped and the working electrode is located in the arc; and connecting the working electrode lead, the reference electrode lead and the counter electrode lead to corresponding wiring points of the in-situ electrolytic cell by using conductive silver paint, and connecting the leads to an electrochemical workstation.

3. The in-situ test method for quantitatively detecting adhesion behavior of single living bacteria according to claim 1, wherein the counter electrode and the working electrode are obtained by evaporating a layer of titanium on the bottom surface of the tank and then evaporating a layer of gold on the titanium layer; the reference electrode is a silver wire with a silver chloride layer plated on the surface, then the silver wire plated with the silver chloride layer is fixed on the wall of the in-situ electrolytic cell, the silver wire plated with the silver chloride layer is ensured to be arranged in the cell, and the exposed silver wire is arranged outside the cell and is used as a reference electrode wiring point.

4. The in situ test method for quantitatively detecting adhesion behavior of a single living bacterium according to claim 1, wherein an annular silica gel pad is disposed between the upper edge of the environmental chamber and the AFM base plate, and between the lower edge thereof and the stage of the optical inverted microscope, thereby forming a sealed environment inside the environmental chamber for maintaining different test environments.

5. The in situ test method for quantitatively detecting adhesion behavior of a single living bacterium of claim 1, wherein the central circular aperture of the optical inverted microscope stage is sealed with a cover slip.

6. The in situ test method for quantitative detection of adhesion behavior of a single living bacterium according to claim 1, wherein the movement of the sample stage in up and down, left and right, front and back directions is specifically: a X, Y direction adjusting connecting rod and a magnetic lifting connecting rod are arranged below the AFM base plate, and a X, Y direction positioning hole for inserting the X, Y direction adjusting connecting rod is formed in the sample table; the sample platform is made of stainless steel, and the lifting connecting rod with magnetism can absorb the upper surface of the stainless steel sample platform so as to fix the sample platform.

7. The in situ test method for quantitatively detecting adhesion behavior of a single living bacterium of claim 1, wherein the shock absorbing stage is suspended in an electromagnetic shielding box by four elastic bands.

8. The in situ test method for quantitatively detecting adhesion behavior of a single living bacterium according to claim 1, wherein the shielding case is provided with a sound absorbing material on an inner surface thereof for eliminating influence of environmental noise on a test process.