Bacterial biofilm metabolic activity detection method based on scanning electrochemical microscope technology and microarray

Abstract

The invention belongs to the technical field of scanning electrochemical microscopes, and discloses a bacterial biofilm metabolic activity detection method based on a scanning electrochemical microscope technology and a microarray, and the method comprises the following steps: inoculating bacteria to be detected into microarray holes, applying an antibacterial agent for incubation, the method comprises the following steps: adding a phosphate buffer solution containing resazurin, ferrocene methanol and glucose for determination, positioning an SECM probe to a fixed height position on the surface of a microarray, and carrying out global scanning on the whole microarray in a line scanning manner to obtain a reduction current signal measured by an electrode. According to the method, the metabolic activity of the biological membrane can be accurately indicated, and misjudgment and background color interference generated when an optical signal of a resazurin reduction product is used for detection can be avoided. The metabolic activity change of the biological membrane before and after antibacterial agent treatment can be researched and represented so as to assist in screening and development of the antibacterial agent, and the method can also be used for biological membrane pollution detection in the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of scanning electrochemical microscopy, and particularly relates to a method for detecting the metabolic activity of bacterial biofilms. Background Art

[0002] Biofilms are structured communities composed of bacteria and their extracellular matrix, and are often attached to biological and non-biological surfaces. They are ubiquitous in natural and human living environments and are of great significance in many fields such as environmental science and biomedicine. Biofilms can be used for sewage treatment and environmental remediation, but they are also the root cause of various difficult-to-cure chronic infections and industrial biofouling. Surfaces contaminated with biofilms (such as drinking water delivery systems, food, and medical devices) can become reservoirs of pathogenic bacteria, continuously spreading pathogenic bacteria to the outside world. In addition, due to their structural heterogeneity and complex physiological processes (such as cell-to-cell communication), biofilms have much higher tolerance to antibacterial agents (antibiotics, disinfectants, or fungicides) than free bacteria. Therefore, the response characteristics of planktonic bacteria to antibacterial agents do not necessarily represent the actual state of bacteria in biofilms. In order to screen and develop effective antibacterial agents that can eliminate biofilms, there is an urgent need to develop new strategies to study and characterize the changes in the metabolic activity of biofilms before and after treatment with antibacterial agents.

[0003] In the past few decades, a variety of methods have been developed to evaluate the effect of antibacterial agents on biofilms. Commercial methods for biofilm analysis mainly rely on colony-forming unit counting or colorimetric assays, and the commercially available method with relatively high recognition is the LIVE / DEAD biofilm viability kit from Invitrogen. Although these methods can analyze biofilm formation or their sensitivity to antibacterial agents, they are usually destructive and require staining or dispersing the biofilm for visualization and quantitative analysis, making the measurement results not necessarily consistent with the actual physiological state in the biofilm. Although the LIVE / DEAD biofilm viability kit from Invitrogen is commonly used in scientific research, it can only distinguish between live and dead cells and does not distinguish the strength of metabolic activity. Therefore, developing a method that can achieve non-destructive analysis of biofilms and real-time in-situ monitoring of the strength of biofilm metabolic activity is of great significance for accurately understanding the response of biofilms to antibacterial agents.

[0004] Scanning electrochemical microscopy (SECM) shows great potential in the non-destructive analysis and quantitative detection of metabolites in biofilms. During biological metabolism, by scanning a microelectrode over a sample immersed in a specific electrolyte solution, high spatiotemporal resolution characterization of different redox-active molecules can be achieved in a targeted manner. The application of SECM in biofilm research mainly focuses on studying electroactive metabolites, quorum-sensing signal molecules, nutrient and oxygen consumption, ion concentration, and pH value. Since the electrode does not contact the sample surface and the measurement environment has good biocompatibility, SECM can achieve non-destructive and real-time in-situ monitoring of biofilms. In addition, compared with the aforementioned optical colorimetric methods, it also has the advantage of avoiding background color interference because it does not rely on optical signals but detects electrical signals. Although methods for monitoring extracellular electron transfer (EET), glucose consumption, or metabolite accumulation have been developed based on SECM to evaluate the metabolic activity of biofilms under specific conditions, these methods all have their limitations. For example, EET only exists in specific bacterial species and is not applicable to all organisms; due to metabolic heterogeneity within bacterial biofilms and between different species, glucose consumption and metabolite accumulation are usually used to study the intensity of specific metabolic behaviors rather than representing overall metabolic activity. Therefore, it is necessary to develop a SECM method that can accurately evaluate the overall metabolic activity of all types of bacterial biofilms. Summary of the Invention

[0005] The object of the present invention is to develop a non-destructive and high-throughput in-situ analysis method for analyzing the metabolic activity of biofilms based on scanning electrochemical microscopy (SECM), which can monitor and evaluate the influence of antibacterial agents or other substances on the metabolic activity of biofilms without destroying the heterogeneous structure of the biofilms.

[0006] To solve the above technical problems, the technical solution proposed by the present invention is as follows: A method for detecting the metabolic activity of bacterial biofilms based on scanning electrochemical microscopy technology and microarray, comprising the following steps: (1) Inoculate the bacteria to be tested into the microarray wells to obtain a coagulated and aggregated bacterial biofilm microarray, apply an antibacterial agent to the microarray, and incubate. (2) Place the microarray incubated in step (1) into a reaction vessel, add a phosphate buffer solution containing resazurin, ferrocenemethanol, and glucose as the measurement solution, immerse the microarray, and immerse the SECM probe (working electrode), reference electrode, and counter electrode in the measurement solution for measurement; wherein resazurin serves as a redox mediator for detecting the metabolic activity of the biofilm, ferrocenemethanol serves as a redox mediator for positioning the probe, and glucose provides a carbon source for the biofilm during the measurement process. (3)Position the SECM probe at a fixed height on the surface of the microarray and perform a global scan of the entire microarray in a line scan mode to obtain the reduction current signal measured by the electrode, thus completing the high-throughput detection of the metabolic activity of the bacterial biofilm.

[0007] Resazurin is a small molecular weight dye that is commonly used as an indicator for optical colorimetric determination and also has electrochemical activity. It can penetrate cell membranes and be sequentially reduced to resorufin and dihydroresorufin by several different reductases (such as NADH, NADPH, and FADH) in living cells. These reduction products can diffuse from inside the cells into the surrounding medium. Among them, resorufin is a strongly fluorescent pink compound that can be detected by colorimetric or fluorescence techniques. The reaction of resazurin being reduced to dihydroresorufin by cells is commonly used to indicate the metabolic activity of various eukaryotic and prokaryotic cells. However, the conversion between these three substances is not a simple irreversible reaction. In two consecutive two-electron reduction reactions, the weakly fluorescent resazurin first receives two electrons and is irreversibly reduced to the strongly fluorescent resorufin. On this basis, it further receives two electrons and is reduced to non-fluorescent dihydroresorufin. The second reduction reaction is reversible under aerobic conditions, and dihydroresorufin can be re-oxidized to resorufin. In this case, the measured optical signal (absorbance or fluorescence value) is not only attributed to the metabolic activity of the cells but also affected by the oxygen concentration, which can lead to an overestimation of the cell metabolic activity. In addition, the second reaction is irreversible under anaerobic conditions. When the reaction is relatively intense, it will weaken the fluorescence intensity to a certain extent, resulting in an underestimation of the cell metabolic activity, especially when measuring high-density cell aggregates with extremely high metabolic activity (such as biofilms or tissues). To ensure as accurate results as possible when using resazurin to detect the metabolic activity of planktonic bacteria, complex condition optimization is required for different bacterial species, bacterial liquid concentrations, culture conditions (aerobic or anaerobic), etc.

[0008] Since resazurin has electrochemical activity, monitoring the consumption of resazurin by bacteria using electrochemical methods rather than measuring the optical signals (absorbance or fluorescence values) of its reduction products can largely overcome the above-mentioned defects, thus achieving more accurate evaluation of metabolic activity. Therefore, some researchers have developed methods for detecting the metabolic activity or drug sensitivity of planktonic bacteria based on the electrochemical activity of resazurin. However, in fact, when resazurin is applied to the detection of planktonic bacteria, even when detecting its electrochemical activity, similar problems encountered in optical detection methods still exist. Since planktonic bacteria are uniformly dispersed in the resazurin solution and their consumption of resazurin persists, the concentration of resazurin continuously decreases. To ensure the comparability of results between different samples, it is necessary to strictly control the reaction time experienced during each measurement to be consistent. In addition, different bacterial concentrations result in different consumption rates of resazurin. For example, if the concentration is too low, it may be difficult to detect distinguishable data, and if the concentration is too high, the reaction will be too fast, making it difficult to ensure the consistency of the measurement time between different samples and rendering the results incomparable. Different bacterial species also have different consumption rates of resazurin. To obtain accurate and comparable results, it is still necessary to optimize the detection conditions (such as bacterial concentration, resazurin concentration, incubation time, etc.) for specific bacterial species. As a result, each type of bacteria requires a set of its own detection parameters during the actual detection process, making the operation process very complex and difficult to truly be applied in practice.

[0009] Interestingly, the present invention finds that when resazurin is combined with SECM for detecting the metabolic activity of bacterial biofilms - rather than planktonic bacteria - it shows unique advantages and the aforementioned problems no longer exist. This is determined by the measurement principle of SECM and the unique physiological characteristics of biofilms. SECM usually measures by immersing a solid sample in a solution with a volume much larger than the sample. The sample reacts with the redox mediator in the solution, causing a stable diffusion layer to form rapidly around the sample. In this diffusion layer, the redox mediator is distributed from the inside out with a certain concentration gradient, and the change trend of the concentration gradient is determined by the inherent reaction rate of the sample. Once the rate is determined, the diffusion layer remains stable. Since the volume of the bulk solution is much larger than the sample, its concentration is always infinitely close to the initial reaction concentration and is considered to be constant in the SECM theoretical system and practical applications. This bulk solution with a constant concentration provides a continuous supply of redox mediators for the reaction to maintain the stability of the diffusion layer. By using the microelectrode probe of SECM with high spatial resolution to detect the concentration of the redox mediator in the diffusion layer, the reaction activity of the sample can be judged. Bacterial biofilms are high-density cell aggregates, which are solid and tangible. Therefore, they are particularly suitable for forming a stable diffusion layer through the reaction occurring in a local small area in the large solution system of SECM for accurate measurement by the microelectrode probe. The huge difference in volume enables the rapid formation and stability of the diffusion layer, so there is no need to consider the influence of the incubation time on the results. Although the gradient of the diffusion layer will change with the concentration of resazurin and the bacterial density, regardless of the high or low concentration or bacterial density, the diffusion layer will exist stably. Therefore, as long as a concentration and a bacterial density are selected and the same parameter value is used in all experiments, the sample can be measured at any time after being put into the solution to obtain comparable results without special optimization, and the detection time window is wide, without worrying about the error caused by different incubation times. The present invention further uses the developed biofilm microarray to provide biofilms with the same initial cell density and the same shape and size for the detection process. The whole process can achieve large-scale detection without complicated condition optimization, and the measurement results have good repeatability and comparability. Currently, no one has developed a similar SECM method using resazurin as a redox mediator to evaluate the overall metabolic activity of such high-density cell aggregates as biofilms.

[0010] The present invention uses resazurin as a redox mediator and monitors the consumption of resazurin around the biofilm through an SECM probe to indicate the metabolic activity of the biofilm. It should be emphasized that this method detects the consumption of resazurin by the biofilm using the reduction current signal of resazurin under a bias voltage. Compared with the traditional optical detection that relies on the fluorescence or absorbance of luminol (the reduction product of resazurin) generated after the biofilm reduces resazurin, it can avoid potential misestimation of the metabolic activity of bacterial biofilms with high cell density characteristics (as mentioned before, the reversible conversion between strongly fluorescent luminol and non-fluorescent hydro-luminol is the root cause of misjudgment). At the same time, a biofilm microarray based on alginate hydrogel has been developed to provide biofilms with consistent morphology for SECM scanning, so that the influence of the morphology of all biofilms on the probe tip current is the same, making the electrical signals comparable between each biofilm, thereby realizing high-throughput monitoring of multiple samples under multiple treatment factors and obtaining batch electrochemical signal data with statistical value, which is of great value for obtaining accurate and practically guiding conclusions. At the same time, since this method is a non-destructive analysis method, it can also monitor the metabolic activity of the same biofilm at different time points, so as to obtain a more accurate time-effect relationship.

[0011] Specifically, by immersing the biofilm in an electrolyte solution containing an appropriate concentration of resazurin, a microelectrode probe is used to detect the consumption of resazurin by the biofilm to judge the metabolic activity of the biofilm, and realize spatial distribution imaging and in-situ real-time monitoring of the metabolic activity. It can be applied to the following fields: drug sensitivity tests of bacterial biofilms in the field of microbial inspection; disinfection effect tests of biofilm removal strategies (disinfectants, disinfection methods) in the field of public health; optimization of the pollutant degradation ability of biofilms in the field of environmental pollution control; identification and monitoring of biotoxic substances in the field of environmental monitoring; optimization of the power generation efficiency of biofilms in the field of bioenergy.

[0012] For the above detection of the metabolic activity of bacterial biofilms, preferably, in step (1), the microarray uses a polydimethylsiloxane thin film as the substrate, and its array micropores are circular.

[0013] Preferably, in step (1), the method for inoculating the bacteria to be tested into the pores of the microarray is as follows: Drop the modification solution into the array micropores of the microarray, dry it, and then mix the bacteria to be tested, the culture medium and sodium alginate and add them into the array micropores for culture.

[0014] Preferably, the modification solution is an aqueous solution containing 0.005% - 0.05% polylysine and 10 - 100 mmol / L barium chloride by mass concentration; before mixing the bacteria to be tested with sodium alginate, resuspend the bacterial solution with the culture medium and control its concentration at OD 600≈ 2.0, and then mix the bacterial solution with an equal volume of sodium alginate with a mass concentration of 2 - 4%; after overnight culture, the bacterial cell density reaches 1 × 10 10 ~ 10 11 cells / mL.

[0015] Preferably, in step (1), the incubation temperature is 25 - 37°C. The incubation time varies depending on the nature of the antimicrobial agent and the detection purpose. The incubation time for antibiotics is 1 h or more, and the incubation time for disinfectants is 10 min or more. Preferably, in step (2), the assay solution contains: 10 - 500 μmol / L resazurin, 0.5 - 1 mmol / L ferrocenemethanol, 10 - 100 mmol / L glucose; the pH of the assay solution is 7.2 - 7.4.

[0016] Preferably, the working electrode is a disk-shaped carbon microelectrode, the reference electrode is an Ag / AgCl electrode, and the counter electrode is a platinum wire electrode.

[0017] Preferably, in step (2), the reaction is carried out in a deoxygenated environment.

[0018] Preferably, in step (3), the preparation method of the SECM probe is as follows: Fix a carbon fiber with a diameter of less than 30 μm on a copper wire with conductive silver glue, dry it, insert it into a glass capillary, pull the electrode, and then heat-seal the tip under a vacuum state to form a probe. Coat the opening of the probe with epoxy resin to fix the joint of the capillary end and the copper wire, and then polish the tip to obtain an SECM probe with an electrode diameter of less than 25 μm and an RG ratio of less than 2.

[0019] Preferably, in step (3), the operation method of globally scanning the entire microarray in a line scan mode is as follows: First, place the SECM probe below the liquid surface of the assay solution, bias the potential at 0.4 - 0.5 V, use ferrocenemethanol as the redox mediator, and use the SECM probe to make an approach curve on the surface of the microarray (the SECM probe moves below the liquid surface, and when the current value of the approach curve shows an inflection point, stop the probe), so as to accurately position the probe on the surface of the microarray; then use a stepper motor to raise the SECM probe to a position 50 - 500 μm away from the surface of the microarray, bias the potential at -0.4 - -0.7 V, use resazurin as the redox mediator, and globally scan the entire microarray in a line scan mode to obtain the reduction current signal generated by the electrode.

[0020] Preferably, in step (3), after the scanning is completed, further select the biofilm of interest for SECM imaging to obtain the spatial distribution information of metabolic activity. The specific operation is as follows: Raise the SECM probe to a height of at least 1500 μm from the microarray, measure the limiting diffusion current of resazurin in the bulk solution, and normalize all the obtained current data with the limiting diffusion current of the bulk solution for plotting the spatial imaging map of the metabolic activity of the bacterial biofilm.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In this method, the reduction current of resazurin is detected to indicate the concentration of resazurin at a specific position (i.e., the selected probe height) in the diffusion layer around the biofilm (the resazurin diffusion layer is generated by the biofilm consuming the nearby resazurin, and the concentration gradient of the diffusion layer is determined by the strength of the biofilm metabolic activity). The difference between this concentration and the bulk concentration is used to characterize the metabolic activity of the biofilm. Since the reduction of resazurin is irreversible, the metabolic activity of the biofilm can be accurately indicated, and the misjudgment and background color (from the weak fluorescence of resazurin) interference generated when detecting using the optical signal of the resazurin reduction product can be avoided.

[0022] (2) In this method, the probe can be accurately positioned at any distance above the biofilm, so as to measure the concentration of resazurin in its diffusion layer. Since the volume of the biofilm is much smaller than the measurement solution in which it is immersed, the resazurin diffusion layer around the biofilm is stable. No matter how long it is immersed or what concentration of resazurin is used, since the metabolic activity of the biofilm is determined and unchanged during the measurement, the concentration of resazurin in the diffusion layer is determined. The signals obtained by measuring any type of bacterial biofilm at any time are consistent, which can effectively ensure the comparability of the results of the same batch of measurements and different batches of measurements. This method for detecting the metabolic activity of high-density cell aggregates (such as biofilms, human or animal tissues) has the advantages of convenient operation, wide detection time window, good repeatability, strong comparability of results between batches and within batches, universal applicability to all bacterial species, and no need for targeted optimization of conditions.

[0023] (3) This method can analyze the heterogeneous distribution of the metabolic activity of biofilms. Since the diameter of the microelectrode probe is much smaller than the size of the biofilm, by scanning the biofilm in the x-y plane or in the z-axis direction using the probe, the spatial distribution information of the biofilm activity can be obtained, and the resolution can be achieved by adjusting the probe diameter and the scanning speed. Compared with the currently most commercialized fluorescence labeling method for living and dead staining of biofilms, due to the difference in principles, this method can obtain more abundant biofilm metabolic activity information; compared with the staining method, this method does not need to destroy the biofilm structure and will not leave the staining background remaining from the previous measurement. Therefore, continuous monitoring at multiple time points can be carried out on the same sample to obtain more abundant and comparable information; the developed SECM based on extracellular electron transfer (EET), glucose consumption or metabolite accumulation mentioned in the technical background is only applicable to the judgment of metabolic activity in specific species or specific situations, while the reaction principle on which this method is based exists in all organisms, so it has universal applicability.

[0024] (4) This method can not only study and characterize the changes in the metabolic activity of biofilms before and after the treatment with antibacterial agents to assist in the screening and development of antibacterial agents, but also be widely used in the detection of biofilm pollution in the environment, such as in multiple application fields including water delivery systems, the food field, sewage treatment and environmental remediation. Brief Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is the detection device and schematic diagram, where: (A) Schematic diagram of the SECM experimental device for measuring the metabolic activity of biofilms in a biofilm microarray in the deoxygenation tank; (B) Schematic diagram of the principle of measuring the metabolic activity of biofilms using resazurin as a redox mediator (Rz: resazurin, Rf: resorufin, DRf: dihydroresorufin); (C) Chemical reaction formula for the step-by-step conversion of resazurin to resorufin and dihydroresorufin.

[0027] Figure 2 It is an optical image of the morphology of a biofilm in a biofilm microarray.

[0028] Figure 3 It is the resazurin reduction current curve obtained by linearly scanning the SECM probe at different heights above three adjacent living biofilms of Pseudomonas aeruginosa.

[0029] Figure 4 It is a schematic diagram of the resazurin diffusion layer around three adjacent biofilms on a microarray immersed in the SECM measurement solution (the legend [Resazurin] in the upper left corner indicates the resazurin concentration).

[0030] Figure 5 It is a representative line scan current curve measured after applying different doses of ciprofloxacin to Pseudomonas aeruginosa biofilms for 4 h in the examples.

[0031] Figure 6 It is a statistical comparison of the normalized reduction current data of each dose group measured after applying different doses of ciprofloxacin to Pseudomonas aeruginosa biofilms for 4 h in the examples (the sample size of each dose group is 3, and the data are expressed as x ± SD). Note: * indicates compared with the 0 μg / mL dose group, p < 0.05, with statistical significance; † indicates compared with the 50 μg / mL dose group, p < 0.05, with statistical significance; # indicates compared with the 500 μg / mL dose group, p < 0.05, with statistical significance; & indicates compared with the 5000 μg / mL dose group, p < 0.05, with statistical significance.

[0032] Figure 7 It is the dose - effect relationship between the ciprofloxacin concentration and the biofilm metabolic activity (expressed as normalized current) measured after applying ciprofloxacin to Pseudomonas aeruginosa biofilms for 4 h in the examples. The red line is the result of logistic fitting.

[0033] Figure 8 It is a comparison of the reduction currents of each dose group monitored at different time points after applying ciprofloxacin to Pseudomonas aeruginosa biofilms in the examples.

[0034] Figure 9 It is a representative SECM imaging measured after applying different concentrations of ciprofloxacin (antibiotic) to Pseudomonas aeruginosa biofilms for 4 h in the examples.

[0035] Figure 10 It is a representative SECM imaging measured after applying different concentrations of benzalkonium bromide (disinfectant) to Pseudomonas aeruginosa biofilms for 1 h in the examples. Detailed implementation manners

[0036] For the convenience of understanding the present invention, the following will describe the present invention more comprehensively and meticulously in conjunction with the accompanying drawings of the specification and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0038] Reagents, materials and instruments used in this embodiment: Ferrocenylmethanol (Sigma-Aldrich, USA), resazurin sodium (MedChemExpress, China), ciprofloxacin, barium chloride, sodium alginate, polylysine (PLL), D-glucose and phosphate buffer powder (Aladdin, China), brain heart infusion broth (Qingdao Haibo, China). Borosilicate glass capillary (Sutter Instrument, USA), carbon fiber (WPI, USA), diamond polishing film (3M, USA), polydimethylsiloxane film (Zhongke Materials, China). Scanning electrochemical microscope (CHI 920D, Shanghai Chenhua), desktop visual spotting instrument (DS331, Xiamen Weitewei).

[0039] Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0040] The present invention uses the prepared microelectrode with a diameter of less than 25 μm as the SECM probe, immerses the prepared biofilm microarray in a phosphate buffer solution containing resazurin and ferrocenylmethanol with a volume much larger than that of each biofilm, and a resazurin diffusion layer with a stable concentration gradient will be rapidly formed around the biofilm. The concentration gradient is determined by the metabolic activity of the biofilm. First, ferrocenylmethanol is used as the redox mediator, the potential is biased at 0.4 V, and the approach curve of the probe is made through the negative feedback mode to position the probe at a fixed height above the biofilm microarray. Then, the potential is biased at -0.6 V to make the probe competitively reduce resazurin with the biofilm, and the center lines of each row of the array are scanned and measured to obtain the reduction current (linearly related to the resazurin concentration) of each well, representing the metabolic activity level of each well. The device and principle are shown in Figure 1 At a fixed height above the specific well position, an x-y plane scan is performed to obtain a dataset of reduction currents and draw a spatial distribution image of the biofilm metabolic activity.

[0041] The specific technical solution is as follows: (1) Preparation of SECM probe: The carbon fibers (with a diameter of 30 μm or less, usually 30 μm, 10 μm, or 7 μm) are cut into 1-cm lengths, fixed onto copper wires with conductive silver glue, and after drying, inserted into glass capillaries. Electrodes are pulled using a horizontal puller (brand: Sutter Instrument, model: P-97), and the pulling program is: pressure = 500, temperature = 545, stretch = 80, speed = 70, time = 250. The pulled electrodes are further heated and sealed at the tip in a vacuum using a vertical puller (brand: Narishige, model: PC-100). Epoxy resin is applied at the opening of the probe to fix the joint between the capillary end and the copper wire. Then the tip is polished vertically using a needle grinder to obtain a probe with an electrode diameter of less than 25 μm and an RG ratio (the ratio of the diameter of the electrode tip including the glass shell to the diameter of the conductive carbon fiber at the center of the electrode) of less than 2.

[0042] (2) Preparation of the bacterial biofilm microarray: A polydimethylsiloxane film with a thickness of 500 μm is taken, and circular array microholes (500 μm thick, 500 μm in diameter, 800 μm in spacing) are punched using an array puncher. After cleaning and drying, it is pasted onto a sterile glass slide. A modifier solution (an aqueous solution containing 0.005% polylysine and 25 mmol / L barium chloride) is spotted into the array holes using a spotter, 100 nL per hole, and then placed in a biosafety cabinet to dry for later use.

[0043] The bacterial liquid cultured in a liquid medium with shaking for 24 h is centrifuged and washed, and the concentration of the bacterial liquid is adjusted with a 6-fold concentration of the medium to make OD 600 ≈ 2.0, and then it is mixed evenly with an equal volume of 4% sodium alginate, loaded into a spotter, and spotted into the microarray holes prepared in the previous step, 100 nL per hole. During the spotting process, the humidity in the operating room of the spotter is maintained at 80 - 90%. The sodium alginate containing bacteria spotted into the holes will quickly solidify into a semi-solid, which is consistent with the characteristics of a naturally formed biofilm. After the spotted microarray is placed in a wet box and cultured at 37 °C for 1 day, the bacterial cell density in the biofilm reaches 1.6 × 10 11 cells / mL, which is consistent with the cell density of a naturally formed biofilm. Different concentrations of antibacterial agents can then be applied, and the changes in the metabolic activity of the biofilm after different action times are monitored using SECM.

[0044] (3) Monitoring the metabolic activity of the biofilm using SECM: The solution used for SECM measurement is a phosphate buffer solution (pH 7.4) containing 50 μmol / L resazurin, 1 mmol / L ferrocenemethanol, and 10 mmol / L glucose. The measurement process is carried out in an anaerobic environment (because there are two states of aerobic and anaerobic respiration in the bacteria in the biofilm, and by removing oxygen, the respiration state of all bacteria is switched to the metabolic mode of anaerobic respiration, making the results more comparable). After rinsing the biofilm microarray with PBS to remove the applied antibacterial agent, it is fixed to the bottom of the petri dish, and the SECM measurement solution is added and immersed. The carbon microelectrode prepared in step (1) is used as the working electrode, the Ag / AgCl (containing 3M KCl) electrode is used as the reference electrode, and the platinum wire is used as the counter electrode. First, the potential is biased at 0.4 V, and an approach curve is made with ferrocenemethanol as the redox mediator to position the probe 200 μm away from the surface of the microarray. Then, the potential is biased at -0.6 V, and resazurin is used as the redox mediator to make the electrode competitively reduce resazurin in the diffusion layer around the biofilm with the biofilm, and the reduction current signal generated by the electrode indicates the metabolic activity of the biofilm. When the entire array is globally scanned in a line scan mode, the line scan is carried out along the x-axis, the scanning speed is 30 μm / s, the step distance is 3 μm, and the sampling time interval is 0.1 s. When SECM imaging is performed on a specific biofilm, the scanning range is 600 × 600 μm 2 , the scanning speeds in the x-axis and y-axis directions are both 30 μm / s, and the sampling time interval is 1 s. Finally, the probe is raised to a height of at least 1500 μm from the substrate to measure the limiting diffusion current of resazurin in the bulk solution. The obtained current data are all normalized by the limiting diffusion current of the bulk solution for comparison.

[0045] Example: A method for detecting the metabolic activity of bacterial biofilms based on scanning electrochemical microscopy technology and microarrays, comprising the following steps: 1. Preparation of the SECM probe Cut carbon fibers (with a diameter of 30 μm or less, usually 30 μm, 10 μm or 7 μm) into 1 cm lengths, fix them on copper wires with conductive silver glue, and after drying, insert them into glass capillaries. Use a horizontal needle puller (brand: Sutter Instrument, model: P-97) to pull the electrodes. The pulling procedure is: pressure = 500, temperature = 545, stretch = 80, speed = 70, time = 250. The electrodes obtained by pulling are further heated and sealed at the tip in a vacuum state using a vertical needle puller (brand: Narishige, model: PC-100). Apply epoxy resin at the opening of the probe to fix the joint between the capillary end and the copper wire. Then vertically polish the tip with a needle grinder to obtain a probe with an electrode diameter of less than 25 μm and an RG ratio (the ratio of the diameter of the electrode tip including the glass shell to the diameter of the conductive carbon fiber at the center of the electrode) of less than 2.

[0046] 2. Preparation of bacterial biofilm microarrays and application of treatment factors Take a polydimethylsiloxane film with a thickness of 500 μm, use an array punch to punch out circular array microholes (500 μm thick, 500 μm in diameter, 800 μm spacing), clean and dry it, and then attach it to a sterile glass slide. Use a spotter to spot the modification solution (an aqueous solution containing 0.005% polylysine and 25 mmol / L barium chloride) into the array holes, 100 nL per hole, and then place it in a biosafety cabinet to dry for later use.

[0047] After centrifuging and washing the bacterial liquid that has been shaken and cultured in liquid medium for 24 h, adjust the concentration of the bacterial liquid with a 6-fold concentration of the medium to make OD 600 ≈ 2.0, then mix it evenly with an equal volume of 4% sodium alginate, load it into a spotter, and use the spotter to spot it into the microarray holes prepared in the previous step, 100 nL per hole. Keep the humidity in the operating room of the spotter at 80 - 90% during the spotting process. The sodium alginate containing bacteria in the holes will quickly solidify into a semi-solid, and its shape is a hemispherical shape protruding from the plane of the polydimethylsiloxane film ( Figure 2 ), and the surface topography has a slight granularity (i.e., slight roughness, not absolutely smooth), which is caused by small clusters of bacteria aggregating.

[0048] After culturing the spotted microarray in a humid box at 37 °C for 1 day, different concentrations of antibacterial agents can be applied to the biofilms on the microarray. The antibacterial agents are spotted onto each well of the microarray in a non-contact spotting manner using a spotter, and at least three parallel wells are applied for each dose to obtain statistically significant data. Specifically, if the antibacterial agent is an antibiotic (ciprofloxacin is taken as an example in this embodiment), its killing process for biofilms is slow, and the action time span is from several hours to several days. Therefore, before performing SECM detection, the applied antibiotic is directly rinsed with PBS and then the detection can be carried out. If the antibacterial agent is a disinfectant (benzalkonium bromide is taken as an example in this embodiment), its killing process is rapid, and the action time span is from several minutes to dozens of minutes. Therefore, before performing SECM detection, the residual disinfectant needs to be neutralized with the corresponding neutralizer, and then the neutralizer is rinsed off with PBS before detection.

[0049] (3)SECM monitoring of biofilm metabolic activity 1) Assemble the measurement system: The solution used for SECM measurement is a phosphate buffer solution (pH 7.4) containing 50 μmol / L resazurin, 1 mmol / L ferrocenemethanol, and 10 mmol / L glucose. The measurement process is carried out in an oxygen-free environment. The biofilm microarray treated with the antibacterial agent in step (2) is fixed to the bottom of a petri dish using a high-viscosity polydimethylsiloxane film, the SECM measurement solution is added, the microarray is immersed, and the reference electrode and the counter electrode are immersed in the solution. The carbon microelectrode prepared in step (1) is used as the working electrode, the Ag / AgCl (containing 3M KCl) electrode is used as the reference electrode, and the platinum wire is used as the counter electrode.

[0050] 2) Locate the probe height: First, place the probe below the liquid surface and above the polydimethylsiloxane film substrate. Then, the potential is biased at 0.4 V, and ferrocenemethanol is used as the redox mediator. The probe makes an approach curve on the surface of the polydimethylsiloxane film to position the probe on the surface of the microarray, and then the probe is raised to 200 μm above the surface of the microarray. The probe height varies depending on the pore size of the biofilm microarray. In this embodiment, a biofilm with a diameter of 500 μm is used. After measuring the resazurin concentration distribution profiles at different heights of three adjacent identical biofilms using the line scan method in step 3) below, the height at which the current signal is not affected by the small variations in the surface topography of the biofilm, the current curves of the three holes are smooth, and the coefficient of variation of the lowest point current value is less than 5% is determined as the optimal scanning height. In this embodiment, it is 200 μm, and the selection basis is shown in Figure 3 . Figure 3The reduction current curves are obtained by scanning the x-axis direction at different heights above three adjacent biofilms in the microarray. The reduction current decreases as the probe height decreases, indicating that the concentration of resazurin is lower in the area closer to the biofilm surface. The black curve in the figure shows that when the scanning height is 100 μm, the current curve presents irregular sawtooth fluctuations at the trough, which is the sensitive feedback current response of the probe tip to the uneven biofilm surface. As the scanning height increases, this phenomenon gradually disappears, as shown by the red, blue, and green curves in the figure. The trough is smooth, indicating that the slight morphological fluctuations on the biofilm surface no longer contribute to the current signal. The current size at this time depends entirely on the consumption of resazurin by the biofilm. Therefore, when the scanning height is greater than or equal to 200 μm, the current size can be used to accurately evaluate the metabolic activity of the biofilm. Considering that the closer to the biofilm, the higher the contrast between the current and the baseline, the optimal scanning height is 200 μm. Figure 4 For combination Figure 3 Results A schematic diagram of the resazurin diffusion layer (i.e., resazurin concentration distribution) around the biofilm was drawn. The apparent thickness of the diffusion layer was about 1000 μm, and the closer to the biofilm, the lower the resazurin concentration. The size of the diffusion layer is related to the size of the biofilm. Depending on the specific research needs, the biofilm diameter may need to be changed. Once the diameter size is selected, only one resazurin concentration distribution profile scan is required to determine the optimal scanning height for subsequent measurements.

[0051] 3) High-throughput linear scanning and metabolic activity imaging of biofilm metabolic activity The potential was biased to -0.6 V, and resazurin was used as the redox medium. The electrode and the biofilm competed to reduce the resazurin in the diffusion layer around the biofilm, and the reduction current signal generated by the electrode indicated the metabolic activity of the biofilm. When the entire array was globally scanned in a line scan mode, the probe was positioned above the center line of each row of biofilm, and each row of biofilm was line scanned along the x-axis. The scanning speed was 30 μm / s, the step distance was 3 μm, and the sampling time interval was 0.1 s. After the scan was completed, the probe was raised to a height of at least 1500 μm from the substrate, and the limiting diffusion current of resazurin in the bulk solution was measured. The current data obtained were normalized with the limiting diffusion current of the bulk solution (that is, the measured current was divided by the limiting diffusion current of the bulk solution, and the value obtained was the normalized current) for comparison ( Figure 5 The normalized current can be used for statistical analysis between different treatment doses ( Figure 6 ), and obtain the dose-effect relationship (whether there is a dose-effect relationship and the dose-effect relationship pattern) ( Figure 7 ). After the biofilm array is scanned, the same dose of antimicrobial agent can be applied to the corresponding wells according to the method described in step 2. The biofilm array after different treatment times can be scanned, and the obtained data set can be used to analyze the time effect relationship of the antimicrobial agent (Figure 8 ), thus providing useful information for inferring the action mode and mechanism of the antibacterial agent on the biofilm of specific bacterial species.

[0052] After global linear scanning, biofilms can be randomly selected for spatial imaging of metabolic activity to understand the influence of the antibacterial agent on the spatial distribution of biofilm metabolic activity. With the center of the biofilm as the center, the scanning range is 600 × 600 μm 2 , and the scanning speed of the probe in both the x-axis and y-axis directions is 30 μm / s, and the sampling time interval is 1 s. After the scanning is completed, the probe is lifted to a height of at least 1500 μm from the substrate, and the limiting diffusion current of resazurin in the bulk solution is measured. All the obtained current data are normalized by the limiting diffusion current of the bulk solution and used to draw imaging diagrams ( Figure 9 , Figure 10 ).

[0053] 4) Significance and uses of the obtained line scan and imaging data Specifically, in this example, the Pseudomonas aeruginosa biofilm treated with ciprofloxacin is taken as an example. Figure 9 Figure 4 is the SECM imaging diagram of the Pseudomonas aeruginosa biofilm treated with ciprofloxacin at 4 concentrations (0, 50, 500, 5000 μg / mL). The bluer the color, the greater the reduction current, and the redder the color, the smaller the reduction current. By comparing the color distributions of the imaging diagrams at the four doses, it can be intuitively judged that as the concentration of ciprofloxacin increases, the overall reduction current of resazurin increases, that is, the metabolic activity of the biofilm weakens. The line scan results can more conveniently provide data for quantitative statistical analysis. Figure 5 The 4 current curves in Figure 8 correspond to the line scan current curves on the center lines of the four biofilms in Figure 6, in the figure, * indicates a statistical difference from the control group (0 μg / mL); † indicates a statistical difference from the 50 μg / mL group; # indicates a statistical difference from the 500 μg / mL group; & indicates a statistical difference from the 5000 μg / mL group. It can be seen from the figure that the current differences between the three treatment groups of 50, 500, and 5000 μg / mL and the control group are statistically significant, and there are also statistical differences among the three treatment groups. Thus, the conclusion can be drawn that the increase in ciprofloxacin concentration is related to its more obvious killing effect on Pseudomonas aeruginosa biofilms. Further fitting the line scan data of multiple treatment doses can also explain the dose-response relationship of the antibacterial agent on the biofilm. As Figure 7 , the function fitting of the line scan data of parallel samples (3 for each dose point) at 9 ciprofloxacin treatment dose points revealed that the dose-response relationship between ciprofloxacin concentration and the metabolic activity of Pseudomonas aeruginosa biofilms is a logistic function relationship, indicating that the increase in the killing effect caused by a unit dose of ciprofloxacin is more obvious in the low-dose range and gradually weakens in the high-dose range. Since this method is a non-destructive analysis method, the biofilms on the same array can be continuously monitored at different time points to obtain the time-effect relationship of the antibacterial agent on the biofilm. As Figure 8 , is the normalized reduction current of Pseudomonas aeruginosa biofilms after treatment with different concentrations of ciprofloxacin for different times. It can be observed that the normalized currents of the three treatment groups increase over time, reflecting the corresponding decrease in metabolic activity. After 96 h of treatment with 5000 μg / L ciprofloxacin, the normalized current was observed to be approximately 1, indicating that Pseudomonas aeruginosa biofilms were almost completely killed at such a high dose. It can be inferred from the comparison of the time-effect relationships of different doses that the higher the dose, the shorter the time required to achieve complete killing. This result is of great significance for revealing the mode of action of antibacterial agents and assisting in drug use decisions.

[0054] In addition to visually understanding the activity differences of biofilms after treatment with different doses of antibacterial agents, the imaging data of biofilms are more importantly used to understand the active distribution of biofilms after the action of different antibacterial agents. Figure 9 is the imaging of Pseudomonas aeruginosa biofilms after treatment with different doses of ciprofloxacin (an antibiotic), Figure 10Imaging of Pseudomonas aeruginosa biofilms treated with different doses of benzalkonium bromide (a disinfectant). By comparing the two figures, it can be found that ciprofloxacin uniformly weakens the biofilm activity (the reduction current increases uniformly), and at different treatment doses, the activity is uniformly distributed; after the action of benzalkonium bromide, the daily disinfection dose (2 g / L) can cause aggregative active residual areas in the biofilm, and a dose 10 times the daily use dose (20 g / L) is required to kill all the bacteria in the biofilm. These information can help reveal the different action modes of antibacterial agents on biofilms and suggest the possible antibacterial agent tolerance mechanisms behind them.

Claims

1. A method for detecting bacterial biofilm metabolic activity based on scanning electrochemical microscopy technology and microarray, characterized in that: The steps include: (1) Inoculating the bacteria to be tested into the microarray wells to obtain a coagulated and aggregated bacterial biofilm microarray, applying an antibacterial agent to the microarray, and incubating; (2) placing the microarray incubated in step (1) in a reaction container, adding a phosphate buffer containing resazurin, ferrocene methanol and glucose as a measurement solution, immersing the microarray, and immersing the SECM probe, reference electrode and counter electrode in the measurement solution to perform measurement; (3) Position the SECM probe at a fixed height on the microarray surface, perform a global scan of the entire microarray in a line scan mode, and obtain the reduction current signal measured by the electrode, thereby completing the high-throughput detection of bacterial biofilm metabolic activity.

2. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (1), the microarray is based on a polydimethylsiloxane film, and the array microwells are circular.

3. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (1), the method for inoculating the test bacteria into the wells of the microarray is as follows: the modification liquid is spotted into the array wells of the microarray, and after drying, the test bacteria, culture medium and sodium alginate are mixed and added into the array wells for cultivation.

4. The method for detecting bacterial biofilm metabolic activity according to claim 3, characterized in that: The modification solution is an aqueous solution containing 0.005% to 0.05% poly-lysine and 10 to 100 mmol / L barium chloride; before mixing the bacteria to be tested with sodium alginate, the bacterial solution is resuspended in a culture medium and its concentration is controlled at OD 600 ≈ 2.0, and then the bacterial solution was mixed with an equal volume of sodium alginate with a mass concentration of 2-4%; after overnight culture, the bacterial cell density reached 1 × 10 10 ~ 10 11 Pieces / mL.

5. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (1), the incubation temperature is 25 to 37°C.

6. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (2), the assay solution contains: 10 to 500 μmol / L resazurin, 0.5 to 1 mmol / L ferrocene methanol, and 10 to 100 mmol / L glucose; the pH of the assay solution is 7.2 to 7.

4.

7. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (2), the reaction is carried out in a deoxygenated environment.

8. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (3), the preparation method of the SECM probe is as follows: a carbon fiber with a diameter of less than 30 μm is fixed to a copper wire with a conductive silver glue, and after drying, it is inserted into a glass capillary, an electrode is drawn, and then the tip is sealed by heating under vacuum to form a probe, and epoxy resin is applied to the opening of the probe to fix the joint between the capillary end and the copper wire, and then the tip is polished to obtain a SECM probe with an electrode diameter of less than 25 μm and an RG ratio of less than 2.

9. The method for detecting bacterial biofilm metabolic activity according to claim 1, characterized in that: In step (3), the operation method of performing global scanning of the entire microarray in a line scanning manner is as follows: first, the SECM probe is placed below the liquid surface of the assay solution, the potential is biased at 0.4 to 0.5 V, ferrocene methanol is used as a redox medium, and the SECM probe is used to make an approximation curve to the microarray surface, thereby accurately positioning the probe on the microarray surface; Then, the SECM probe was raised to a distance of 50 to 500 μm from the microarray surface using a stepper motor, the potential was biased at -0.4 to -0.7 V, and the entire microarray was scanned globally using resazurin as the redox medium to obtain the reduction current signal generated by the electrode.

10. The method for detecting bacterial biofilm metabolic activity according to any one of claims 1 to 9, characterized in that: In step (3), after the scan is completed, the biofilm is further selected for SECM imaging to obtain the spatial distribution information of metabolic activity; the specific operation is as follows: the SECM probe is raised to a height of at least 1500 μm from the microarray, and the limiting diffusion current of the resazurin in the bulk solution is measured. The current data obtained are normalized with the limiting diffusion current of the bulk solution and used to draw a spatial imaging diagram of the metabolic activity of the bacterial biofilm.

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